JP7277370B2 - Veterinary anti-IL-31 antibody - Google Patents

Description

This application claims the benefit of International Application No. PCT/US2017/023788, filed March 23, 2017, each of which is incorporated herein by reference in its entirety for all purposes; It claims the benefit of US Provisional Patent Application No. 62/463,543 filed on 24th.

The present invention provides isolated anti-IL31 antibodies that bind, e.g., canine IL31, and methods of using them, e.g., to treat IL31-induced conditions in companion animals such as dogs, cats, and horses, or to treat IL31 in cells. It relates to a method of reducing signaling function.

Interleukin 31 (IL31) is a cytokine produced primarily by Th2 cells and is understood to be involved in promoting skin diseases such as pruritus and other forms of allergic disease (e.g. atopic dermatitis). there is IL31 functions by binding its receptor and activating downstream activities, such as activation of JAK1, and is thought to cause many of the clinical problems associated with dermatitis and other disorders.

Companion animals such as cats, dogs, and horses suffer from many skin diseases similar to those in humans, such as atopic dermatitis. However, the IL31 sequence differs between humans, cats, dogs and horses. Accordingly, there is a need for methods and compounds that can be used specifically to bind IL31 in a companion animal to treat IL31-induced conditions and reduce IL31 signaling.

In some embodiments, an isolated antibody that binds canine IL31 is provided. In some embodiments, the antibody binds to an epitope comprising amino acids 34-50 of SEQ ID NO:22. In some embodiments, the antibody binds to an epitope comprising the amino acid sequence of SEQ ID NO:23. In some embodiments, the antibody binds to an epitope comprising the amino acid sequence of PSDX ₁ X ₂ KI (SEQ ID NO: 45), where X is any amino acid residue. In some embodiments, X ₁ is a hydrophobic amino acid. In some embodiments, X ₁ is selected from A, V, I, and L. In some embodiments, _X1 is selected from V and I. In some embodiments, _X2 is a hydrophilic amino acid. In some embodiments, _X2 is selected from A, R, K, Q, and N. In some embodiments, _X2 is selected from R and Q. In some embodiments, X ₁ is V and X ₂ is R. In some embodiments, X ₁ is I and X ₂ is Q. In some embodiments, the antibody binds to an epitope comprising the amino acid sequence of SEQ ID NO:88.

In some embodiments, the antibody is less than 5×10 ⁻⁶ M, less than 1×10 −6 M, less than 5×10 ⁻⁷ M, less than 1×10 ⁻⁷ M, less than 5× ^{10 −6} M as measured by biolayer interferometry. Less than ×10 ⁻⁸ M, less than 1×10 ⁻⁸ M, less than 5×10 ⁻⁹ M, less than 1×10 ⁻⁹ M, less than 5×10 ⁻¹⁰ M, less than 1×10 ⁻¹⁰ M, 5×10 Binds canine IL31 with a dissociation constant (Kd) of less than ⁻¹¹ M, less than 1×10 ⁻¹¹ M, less than 5×10 ⁻¹² M, or less than 1×10 ⁻¹² M.

In some embodiments, the antibody reduces IL31 signaling function in a companion animal species as measured by reduced phosphorylation of STAT-3. In some embodiments, the companion animal species is canine, feline, or equine.

In some embodiments, the antibody binds to feline IL31 or equine IL31 as determined by immunoblot analysis and/or biolayer interferometry. In some embodiments, the antibody competes with a monoclonal M14 antibody for binding to canine IL31. In some embodiments, the antibody competes with the monoclonal M14 antibody for binding to feline IL31. In some embodiments, the antibody does not bind human IL31 as determined by immunoblot analysis and/or biolayer interferometry.

In some embodiments the antibody is a monoclonal antibody. In some embodiments, the antibody is a canine, canine, feline, feline, equine, equine, or chimeric antibody. In some embodiments, the antibody is a chimeric antibody comprising a murine variable heavy chain framework region or a murine variable light chain framework region.

In some embodiments, an antibody comprises a heavy chain and a light chain,
a. the heavy chain has a CDR-H1 sequence that has at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:1; A CDR-having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 2, 62, 89, or 87 an H2 sequence and a CDR-H3 sequence having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:3 including
b. A light chain has a CDR that has at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:63 - a CDR-L2 sequence that has at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the L1 sequence, the amino acid sequence of SEQ ID NO:9; and CDR-L3 sequences having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody has (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2 or 89, and (c) the amino acid sequence of SEQ ID NO:3. A heavy chain comprising a CDR-H3 containing sequence is included.

In some embodiments, the antibody has (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:62 or 87, and (c) an amino acid sequence of SEQ ID NO:3. A heavy chain comprising a CDR-H3 containing sequence is included.

In some embodiments, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and (c) the amino acid sequence of SEQ ID NO:10. A light chain comprising a CDR-L3 comprising

In some embodiments, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:63, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and (c) the amino acid sequence of SEQ ID NO:10. A light chain comprising a CDR-L3 comprising

In some embodiments, the antibody has (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO: 4, 70, or 79, (b) HC-FR2 of SEQ ID NO: 5, 71, or 80 (c) the HC-FR3 sequence of SEQ ID NO: 6, 72, 73, or 81; (d) the HC-FR4 sequence of SEQ ID NO: 7, 74, or 82; (e) the HC-FR4 sequence of SEQ ID NO: 11, 75, or 83 the variable region light chain framework 1 (LC-FR1) sequence, (f) the LC-FR2 sequence of SEQ ID NO: 12, 76, or 84, (g) the LC-FR3 sequence of SEQ ID NO: 13, 77, or 85, or ( h) comprising one or more of the LC-FR4 sequences of SEQ ID NOs: 14, 78, or 86;

In some embodiments, the antibody is
a. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:24; (ii) at least 85% with the amino acid sequence of SEQ ID NO:25; , a variable heavy chain sequence having at least 90%, at least 95%, or at least 98% sequence identity, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or b. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:16; (ii) at least 85% with the amino acid sequence of SEQ ID NO:15; , a variable heavy chain sequence having at least 90%, at least 95%, or at least 98% sequence identity, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or c. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:32; (ii) at least 85% with the amino acid sequence of SEQ ID NO:33; , a variable heavy chain sequence having at least 90%, at least 95%, or at least 98% sequence identity, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii).

In some embodiments, the antibody comprises the variable light chain sequence of SEQ ID NO:24, SEQ ID NO:16, or SEQ ID NO:32. In some embodiments, the antibody comprises the variable heavy chain sequence of SEQ ID NO:25, SEQ ID NO:15, or SEQ ID NO:33. In some embodiments, the antibody comprises the variable light chain sequence of SEQ ID NO:24 and the variable heavy chain sequence of SEQ ID NO:25, the variable light chain sequence of SEQ ID NO:16 and the variable heavy chain sequence of SEQ ID NO:15, or the It contains a variable light chain sequence and a variable heavy chain sequence of SEQ ID NO:33.

In some embodiments, the antibody is a chimeric antibody comprising constant heavy or light chain regions derived from a companion animal.

In some embodiments, the antibody comprises (a) a canine heavy chain constant region selected from IgG-A, IgG-B, IgG-C, and IgG-D constant regions, (b) IgG1, IgG2a, and IgG2b constant regions or (c) a horse heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 constant regions.

In some embodiments, the antibody is
a. (i) the light chain amino acid sequence of SEQ ID NO:26, (ii) the heavy chain amino acid sequence of SEQ ID NO:27, or (iii) the light chain amino acid sequence of (i) and the heavy chain amino acid sequence of (ii), or b. (i) the light chain amino acid sequence of SEQ ID NO:30, (ii) the heavy chain amino acid sequence of SEQ ID NO:31, or (iii) the light chain amino acid sequence of (i) and the heavy chain amino acid sequence of (ii), or c. (i) the light chain amino acid sequence of SEQ ID NO:34, (ii) the heavy chain amino acid sequence of SEQ ID NO:35, or (iii) the light chain amino acid sequence of (i) and the heavy chain amino acid sequence of (ii).

In some embodiments, the antibody comprises the light chain amino acid sequence of SEQ ID NO:21. In some embodiments, the antibody comprises the heavy chain amino acid sequence of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

In some embodiments, the antibody is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab') ₂ and Fab'-SH.

In some embodiments, the antibody is bispecific and the antibody is directed to IL31 and IL17, TNFα, CD20, CD19, CD25, IL4, IL13, IL23, IgE, CD11α, IL6R, α4-integrin, IL12, IL1β , or BlyS.

In some embodiments, isolated nucleic acids encoding the anti-IL31 antibodies described herein above are provided. In some embodiments, host cells are provided that contain nucleic acids encoding the anti-IL31 antibodies described herein above. In some embodiments, producing an anti-IL31 antibody, comprising culturing such a host cell comprising a nucleic acid encoding an anti-IL31 antibody as described herein above and isolating the antibody A method is provided.

In some embodiments, pharmaceutical compositions are provided comprising an anti-IL31 antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, pharmaceutical compositions are provided comprising an anti-IL31 antibody described herein and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is L-histidine, sodium chloride, and Contains polysorbate 80.

In some embodiments, the pharmaceutical composition has a pH of 5.0-6.2. In some embodiments, the pharmaceutical composition has a pH of 5.0-6.0, or 5.3-5.7, or 5.5.

In some embodiments, the pharmaceutical composition has an L-histidine concentration of 5 mM to 100 mM, 10 mM to 50 mM, 20 mM to 30 mM, 10-30 mM, or 20 mM.

In some embodiments, the pharmaceutical composition has a sodium chloride concentration of 80-200 mM, 100-175 mM, 120-150 mM, or 140 mM.

In some embodiments, the pharmaceutical composition has a concentration of polysorbate 80 of 0.005 mg/mL to 0.5 mg/mL, 0.01 mg/mL to 0.1 mg/mL, or 0.05 mg/mL.

In some embodiments, a pharmaceutically acceptable carrier comprises at least one sugar. In some embodiments, the pharmaceutical composition has a concentration of at least one sugar from 0.5% to 20%, 1% to 10%, 1% to 5%, or 1% to 3%. In some embodiments, pharmaceutically acceptable carriers include sucrose, trehalose, D-mannitol, maltose, and/or sorbitol.

In some embodiments, a pharmaceutically acceptable carrier comprises an antimicrobial agent. In some embodiments, the pharmaceutical composition comprises m-cresol or methylparaben. In some embodiments, the pharmaceutical composition comprises 0.2% m-cresol and/or 0.9% methylparaben.

Uses of Antibodies and Pharmaceutical Compositions In some embodiments, a method of treating a companion animal species having an IL31-induced condition comprises a therapeutically effective amount of an anti-IL31 antibody described herein or Methods are provided comprising administering to a companion animal species a pharmaceutical composition comprising the antibodies described herein. In some embodiments, the companion animal species is canine, feline, or equine. In some embodiments, the IL31-induced condition is an pruritus or allergic condition. In some embodiments, the IL31-induced condition is selected from atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, and eczema.

In some embodiments, an anti-IL31 antibody or pharmaceutical composition is administered parenterally. In some embodiments, the anti-IL31 antibody or pharmaceutical composition is administered by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intrasynovial, intrathecal, or inhalation routes. administered.

In some embodiments, the method comprises administering a Jak inhibitor, PI3K inhibitor, AKT inhibitor, or MAPK inhibitor in combination with an anti-IL31 antibody or pharmaceutical composition. In some embodiments, the method combines an anti-IL31 antibody or a pharmaceutical composition with an anti-IL17 antibody, an anti-TNFα antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, an anti-IL13 antibody, an anti-IL23 antibody. administering one or more antibodies selected from antibodies, anti-IgE antibodies, anti-CD11α antibodies, anti-IL6R antibodies, anti-α4-integrin antibodies, anti-IL12 antibodies, anti-IL1β antibodies, and anti-BlyS antibodies.

In some embodiments, a method of reducing IL31 signaling function in a cell, wherein an anti-IL31 antibody or pharmaceutical composition described herein is administered under conditions that allow binding of the antibody to extracellular IL31. exposing a cell to a cell under low temperature conditions, thereby reducing binding to the IL31 receptor and/or reducing IL31 signaling function by the cell. In some embodiments, the cells are exposed to the antibody or pharmaceutical composition ex vivo. In some embodiments, cells are exposed to the antibody or pharmaceutical composition in vivo. In some embodiments, the cells are canine, feline, or equine cells.

In some embodiments, a method of detecting IL31 in a sample from a companion animal species, wherein an anti-IL31 antibody or pharmaceutical composition described herein is used to allow binding of the antibody to IL31 A method is provided comprising contacting a sample under conditions and detecting whether a complex is formed between the antibody and IL31 in the sample. In some embodiments, the sample is a biological sample obtained from a dog, cat, or horse.

Alignment of variable light sequences of M14, M18, M19, and M87 mouse monoclonal antibody clones. Alignment of variable heavy sequences of M14, M18, M19, and M87 mouse monoclonal antibody clones. FIG. 4 is a graph of canine IL31 binding assays with varying concentrations of chimeric M14 antibody. FIG. 4 is a graph of canine IL31 binding assay with varying concentrations of caninized M14 antibody. Immunoblot showing inhibition of canine IL31 signaling with varying concentrations of caninized M14 antibody. Immunoblot of GST-dog-IL31 deletion probed with M14 antibody and anti-GST antibody, respectively. Immunoblot of GST-dog-IL31 deletion probed with M14 antibody and anti-GST antibody, respectively. Immunoblot of feline and equine IL31 proteins fused to human Fc probed with M14 and anti-FC antibodies, respectively. Refine epitope mapping of mature canine IL31 epitopes using anti-canine IL31 antibody (upper panel) and anti-GST antibody (lower panel) and immunoblot analysis of alanine scanning. Refine epitope mapping of mature canine IL31 epitopes using anti-canine IL31 antibody (upper panel) and anti-GST antibody (lower panel) and immunoblot analysis of alanine scanning. Refine epitope mapping of mature canine IL31 epitopes using anti-canine IL31 antibody (upper panel) and anti-GST antibody (lower panel) and immunoblot analysis of alanine scanning. Refine epitope mapping of mature canine IL31 epitopes using anti-canine IL31 antibody (upper panel) and anti-GST antibody (lower panel) and immunoblot analysis of alanine scanning. Refine epitope mapping of mature canine IL31 epitopes using anti-canine IL31 antibody (upper panel) and anti-GST antibody (lower panel) and immunoblot analysis of alanine scanning. Immunoblot cross-reactivity of anti-canine IL31 antibody M14 to walrus IL31.

Description of Specific Sequences Table 1 provides a list of specific sequences referred to herein.

DESCRIPTION OF SPECIFIC EMBODIMENTS Antibodies that bind to canine IL31, feline IL31, or equine IL31 are provided. Also provided are antibody heavy chains and antibody light chains capable of forming antibodies that bind to IL31. Further provided are antibodies, heavy chains and light chains comprising one or more specific complementarity determining regions (CDRs). Polynucleotides encoding antibodies to canine IL31 are provided. Also provided are methods of producing or purifying antibodies to canine IL31. Methods of treatment with antibodies to canine IL31 are provided. Such methods include, but are not limited to, methods of treating IL31-induced conditions in companion animal species. Methods are provided for detecting IL31 in a sample from a companion animal species.

For the convenience of the reader, the following definitions of terms used herein are provided.

As used herein, numerical terms such as Kd are calculated based on scientific measurements and are therefore subject to reasonable measurement error. In some cases, numerical terms may include numbers rounded to the nearest significant digit.

As used herein, "a" or "an" means "at least one" or "one or more," unless otherwise specified. As used herein, the term "or" means "and/or" unless specified otherwise. In the context of multiple dependent claims, the use of "or" when referring back to another claim means only the claims in the alternative.

Anti-IL31 Antibodies Novel antibodies to IL31 are provided, such as antibodies that bind to canine IL31, feline IL31, and/or equine IL31. Anti-IL31 antibodies provided herein include, but are not limited to, monoclonal antibodies, murine antibodies, chimeric antibodies, caninized antibodies, feline antibodies, and equine antibodies. In some embodiments, the anti-IL31 antibody is an isolated murine monoclonal antibody such as M14, M18, M19, and M87.

Monoclonal antibodies M14, M18, M19, and M87 were isolated as follows. Briefly, mice were immunized with canine IL31 and mouse monoclonal antibody clones were obtained by standard hybridoma technology. An enzyme-linked immunosorbent assay (ELISA) was used to screen hybridoma clones producing IL31-binding antibodies. Based on the binding affinity and cell-based functional assays described herein, hybridoma clones producing monoclonal antibodies M14, M18, M19, and M87 were selected for further study. The variable heavy (VH) and variable light (VL) chains of each of the four clones were sequenced and analyzed by sequence alignment (Figure 1).

Also provided herein is the amino acid sequence of monoclonal antibody M14. For example, for monoclonal antibody M14, the variable heavy chain CDRs (SEQ ID NOs: 1-3; SEQ ID NO: 89 is an alternate definition of CDR-H2), the variable light chain CDRs (SEQ ID NOs: 8-10), the variable region heavy chain frame Work sequences (SEQ ID NOs:4-7), and variable region light chain framework sequences (SEQ ID NOs:11-14) are provided. Amino acid sequences of the variable light chain, light chain, variable heavy chain, and variable and hinge heavy chains of monoclonal antibody M14 are provided (SEQ ID NOs: 24, 36, 25, and 40, respectively).

Further provided are the amino acid sequences of the CDRs, framework sequences, variable light chain, variable heavy chain of monoclonal antibodies M18, M19, and M87 (see, eg, FIG. 1). Variable heavy chain CDRs (SEQ ID NOS: 1-3; SEQ ID NO: 89 is an alternate definition of CDR-H2), variable light chain CDRs (SEQ ID NOS: 8-10), variable light chain (SEQ ID NO: 65) for monoclonal antibody M19 , light chain (SEQ ID NO:38), variable and hinge heavy chain (SEQ ID NO:42), and variable heavy chain (SEQ ID NO:68) are provided. Variable heavy chain CDRs (SEQ ID NOS: 1, 62, and 3; SEQ ID NO: 87 is an alternate definition of CDR-H2), variable light chain CDRs (SEQ ID NOS: 63, 9, and 10), variable light chain CDRs (SEQ ID NOS: 63, 9, and 10) for monoclonal antibody M18 Chain (SEQ ID NO:64), light chain (SEQ ID NO:37), variable and hinge heavy chain (SEQ ID NO:41), and variable heavy chain (SEQ ID NO:67) are provided. A variable light chain (SEQ ID NO:66), a light chain (SEQ ID NO:39), a variable and hinge heavy chain (SEQ ID NO:43), and a variable heavy chain (SEQ ID NO:69) are provided for monoclonal antibody M87.

Also provided herein are chimeric, caninized, feline, and equine antibodies derived from monoclonal antibodies M14, M18, M19, and M87. In some embodiments, amino acid sequences of caninized monoclonal antibody M14 are provided, such as SEQ ID NOs: 15-21 and 70-78. In some embodiments, amino acid sequences of feline antibodies derived from monoclonal antibody M14 are provided, such as SEQ ID NOs:32-35 and 79-86. In some embodiments, amino acid sequences of chimeric antibodies derived from monoclonal antibody M14 are provided, such as SEQ ID NOs:26, 27, 30, and 31.

The term "antibody" is used herein in its broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific (bispecific A variety of antibody constructs, including antibody fragments (Fab, F(ab′) ₂ , ScFv, minibodies, diabodies, triabodies, and tetrabodies, etc.), and trispecific antibodies). encompasses The canine, feline, and equine species have different diversity (classes) of antibodies shared by many mammals.

The term "antibody" includes, but is not limited to, fragments capable of binding antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', di-scFv, sdAb (single domain antibody), and (Fab' ) ₂ (including chemically linked F(ab′) ₂ ). Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fab" fragment, whose name reflects its ability to crystallize readily. Fc" fragment is produced. Pepsin treatment yields an F(ab') ₂ fragment that has two antigen-combining sites and still has the ability to cross-link antigen. The term "antibody" also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as murine, human, cynomolgus, canine, feline, equine, and others. Additionally, for all antibody constructs provided herein, variants having sequences from other organisms are also contemplated. Thus, once the murine antibody version is disclosed, one skilled in the art would understand how to convert an antibody based on the murine sequences to feline, canine, equine, or other sequences. Antibody fragments also include single chain scFv, tandem di-scFv, diabodies, tandem tri-sdcFv, minibodies and any other orientation. Antibody fragments also include nanobodies (antibodies with a single monomeric domain without light chains, such as sdAb, a pair of heavy chain variable domains). Antibody fragments, in some embodiments, may also be referred to as being species specific (eg, murine scFv or canine scFv). This refers to the sequence of at least part of the non-CDR regions rather than the source of the construct. In some embodiments, the antibody includes a label or is conjugated to a second moiety.

The terms "label" and "detectable label" refer to a moiety attached to an antibody or its analyte such that a reaction (eg, binding) between members of a particular binding pair is detectable. A labeled member of a particular binding pair is said to be "detectably labeled." Accordingly, the term "labeled binding protein" means a protein that incorporates a label that provides identification of the binding protein. In some embodiments, the label is a detectable marker capable of producing a signal detectable by visual or mechanical means, such as the incorporation of radiolabeled amino acids, or marked avidin (e.g., optical methods or relative binding of biotinylated moieties to polypeptides detectable by fluorescent markers detectable by colorimetric methods or by streptavidin containing enzymatic activity. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionuclides (e.g., ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I , ¹⁷⁷ Lu, ¹⁶⁶ Ho, or ¹⁵³ Sm), chromogens, fluorescent labels (e.g. FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g. horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, Included are predetermined polypeptide epitopes recognized by secondary reporters (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic agents such as gadolinium chelates. Representative examples of commonly employed labels for immunoassays include light-producing moieties such as acridinium compounds and fluorogenic moieties such as fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with yet another moiety.

The term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up the population are identical except for possible naturally occurring variations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. A sample of monoclonal antibodies can therefore bind to the same epitope on the antigen. The modifier "monoclonal" indicates the property of the antibody to be obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or as described in U.S. Pat. No. 4,816,567. It can be made by recombinant DNA methods. Monoclonal antibodies may be isolated, for example, from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554.

In some embodiments, the monoclonal antibody is an isolated murine antibody selected from clones M14, M18, M19, and M87.

"Amino acid sequence" means the sequence of amino acid residues in a peptide or protein. The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may comprise natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition includes both full-length proteins and fragments thereof. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Moreover, for the purposes of this disclosure, a "polypeptide" includes modifications to the native sequence, such as deletions, additions, and substitutions (generally conservative in nature), so long as the protein maintains the desired activity. ) means a protein containing These modifications may be deliberate, such as site-directed mutagenesis, or accidental, such as mutation of the host that produces the protein, or errors due to PCR amplification.

By "IL31" as used herein is meant any native IL31 resulting from the expression and processing of IL31 within a cell. Unless otherwise indicated, the term can be used for any vertebrae including mammals such as primates (e.g. humans and cynomolgus monkeys) and rodents (e.g. mice and rats), and companion animals (e.g. dogs, cats, and horses). Contains IL31 from animal sources. The term also includes naturally occurring variants of IL31, such as splice or allelic variants.

In some embodiments, canine IL31 comprises the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:44. In some embodiments, the feline IL31 comprises the amino acid sequence of SEQ ID NO:28. In some embodiments, the equine IL31 comprises the amino acid sequence of SEQ ID NO:29. In some embodiments, human IL31 comprises the amino acid sequence of SEQ ID NO:46. In some embodiments, the walrus IL31 comprises the amino acid sequence of SEQ ID NO:47. In some embodiments, murine IL31 comprises the amino acid sequence of SEQ ID NO:61. In other embodiments, IL31 is SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or It contains the number 60 amino acid sequence.

The term "IL31 binding domain" of an antibody refers to the binding domain formed by the light and heavy chains of an anti-IL31 antibody that bind IL31.

In some embodiments, the IL31 binding domain binds canine IL31 with greater affinity than it binds human IL31. In some embodiments, the IL31 binding domain binds other companion animal IL31, eg, feline IL31 or equine IL31. In some embodiments, the IL31 binding domain does not bind human IL31.

As used herein, the term "epitope" refers to a target molecule (e.g., a protein, nucleic acid, carbohydrate, or lipid antigen) to which an antigen-binding molecule (e.g., an antibody, antibody fragment, or scaffold protein comprising an antibody-binding region) binds. ) means the upper part. Epitopes often include chemically active surface groupings of molecules such as amino acids, polypeptides, or sugar side chains and have specific three dimensional structural characteristics, as well as specific charge characteristics. Epitopes can be formed from both contiguous or juxtaposed non-contiguous residues (eg, amino acids, nucleotides, saccharides, lipid moieties) of the target molecule. Epitopes formed from contiguous residues (e.g. amino acids, nucleotides, sugars, lipid moieties) are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically Lost by treatment with denaturing solvents. An epitope can include, but is not limited to, at least 3 residues, at least 5 residues, or 8-10 residues (eg, amino acids or nucleotides). In some examples, the epitope is less than 20 residues (eg, amino acids or nucleotides), less than 15 residues, or less than 12 residues in length. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen. In some embodiments, epitopes can be identified by a certain minimal distance to the CDR residues of the antigen binding molecule. In some embodiments, epitopes are identified by the above distances and are further restricted to those residues involved in binding (eg, hydrogen bonding) between antibody and antigen residues. Epitopes can also be identified by various scans, for example, an alanine or arginine scan can reveal one or more residues with which an antigen-binding molecule can interact. Unless explicitly noted, the set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antibody. Rather, the presence of such sets represents a minimal series (or species set) of epitopes. Thus, in some embodiments, the set of residues identified as epitopes represents the minimal epitope associated with the antigen, rather than an exclusive list of residues of the epitope on the antigen.

In some embodiments, the epitope comprises the amino acid sequence PSDX ₁ X ₂ KI (SEQ ID NO: 45), where X is any amino acid residue. In some embodiments, X ₁ is a hydrophobic amino acid. In some embodiments, X ₁ is selected from A, V, I, and L. In some embodiments, _X1 is selected from V and I. In some embodiments, _X2 is a hydrophilic amino acid. In some embodiments, _X2 is selected from A, R, K, Q, and N. In some embodiments, _X2 is selected from R and Q. In some embodiments, X ₁ is V and X ₂ is R. In some embodiments, X ₁ is I and X ₂ is Q. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO:88. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the epitope is within amino acids 34-50 of SEQ ID NO:22. In some embodiments, the epitope comprises amino acids 34-50 of SEQ ID NO:22.

The term "CDR" means a complementarity determining region defined by at least one mode of identification to those skilled in the art. In some embodiments, the CDRs are defined according to any of the Chochia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chochia, the AbM definition, the contact definition, or a combination of the Kabat, Chochia, AbM, or contact definitions. can be done. The various CDRs in the antibody are represented by appropriate numbers and chain types, including but not limited to CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. be able to. The term "CDR" is also used herein to encompass a "hypervariable region" or HVR, which comprises hypervariable loops.

In some embodiments, the anti-IL31 antibody has (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) the amino acid sequence of SEQ ID NO:2, SEQ ID NO:62, SEQ ID NO:89, or SEQ ID NO:87. or (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, the anti-IL31 antibody is (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:63, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, or (c) A light chain comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:10 is included.

In some embodiments, the anti-IL31 antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or 89, and (c) SEQ ID NO: 3 and (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:8 or 63, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and (c) ) a light chain comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the anti-IL31 antibody comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62 or 87, and (c) SEQ ID NO: 3 and (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:8 or 63, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and (c) ) a light chain comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:10.

The term "variable region" as used herein means a region comprising at least three CDRs. In some embodiments, the variable region comprises three CDRs and at least one framework region (“FR”). The terms "heavy chain variable region" or "variable heavy chain" are used interchangeably and refer to a region comprising at least three heavy chain CDRs. The terms "light chain variable region" or "variable light chain" are used interchangeably and refer to a region comprising at least three light chain CDRs. In some embodiments, a variable heavy chain or variable light chain comprises at least one framework region. In some embodiments, the antibody comprises at least one heavy chain framework region selected from HC-FR1, HC-FR2, HC-FR3, and HC-FR4. In some embodiments, the antibody comprises at least one light chain framework region selected from LC-FR1, LC-FR2, LC-FR3, and LC-FR4. Framework regions may be juxtaposed between the light chain CDRs or between the heavy chain CDRs. For example, an antibody has the following structure: (HC-FR1)-(CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3)-(HC-FR4). variable heavy chains with An antibody may comprise a variable heavy chain having the following structure (CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3). Antibodies are variable having the following structure: (LC-FR1)-(CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3)-(LC-FR4) It may also contain a light chain. An antibody may comprise a variable light chain having the following structure (CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3).

In some embodiments, the anti-IL31 antibody comprises (a) the variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO:4, (b) the HC-FR2 sequence of SEQ ID NO:5, (c) SEQ ID NO: (d) HC-FR4 sequence of SEQ ID NO:7; (e) variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO:11; (f) LC-FR2 of SEQ ID NO:12. (g) the LC-FR3 sequence of SEQ ID NO:13; or (h) the LC-FR4 sequence of SEQ ID NO:14. In some embodiments, the anti-IL31 antibody comprises the variable light chain sequence of (a) SEQ ID NO:16, (b) SEQ ID NO:24, or (c) SEQ ID NO:32. In some embodiments, the anti-IL31 antibody comprises the variable heavy chain sequence of (a) SEQ ID NO:15, (b) SEQ ID NO:25, or (c) SEQ ID NO:33. In some embodiments, the anti-IL31 antibody comprises (a) the variable light chain sequence of SEQ ID NO: 16 and the variable heavy chain sequence of SEQ ID NO: 15, (b) the variable light chain sequence of SEQ ID NO: 24 and the variable heavy chain sequence, or (c) the variable light chain sequence of SEQ ID NO:32 and the variable heavy chain sequence of SEQ ID NO:33.

The term "constant region" as used herein means a region comprising at least three constant domains. The terms "heavy chain constant region" or "constant heavy chain" are used interchangeably and refer to a region comprising at least three heavy chain constant domains, CH1, CH2 and CH3. Non-limiting exemplary heavy chain constant regions include γ, δ, α, ε, and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, an antibody containing an α constant region is an IgA antibody, an antibody containing a μ constant region is an IgM antibody, Antibodies containing the ε constant region are IgE antibodies. Specific isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing the γ1 constant region), IgG2 (containing the γ2 constant region), IgG3 (containing the γ3 constant region), IgG4 (containing the γ4 constant region), and IgA Antibodies include, but are not limited to, IgA1 (including α1 constant region) and IgA2 antibodies (including α2 constant region), and IgM antibodies include, but are not limited to, IgM1 and IgM2. The terms "light chain constant region" or "constant light chain" are used interchangeably and refer to the region comprising the light chain constant domain CL. Non-limiting exemplary light chain constant regions include λ and κ. Deletions and changes within the domain that do not alter function are encompassed within the term "constant region" unless otherwise specified. Dogs, cats, and horses have antibody classes such as IgG, IgA, IgD, IgE, and IgM. Among the canine IgG antibody classes are IgG-A, IgG-B, IgG-C, and IgG-D. Among the feline IgG antibody classes are IgG1a, IgG1b, and IgG2. Among the equine IgG antibody classes are IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7.

The term "chimeric antibody" or "chimera" refers to an antibody in which a portion of the heavy or light chain is derived from a particular source or species, while at least a portion of the remainder of the heavy or light chain is derived from a different source or species. means In some embodiments, a chimeric antibody has at least one variable region from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and from a second species (e.g., human, dog, cat, horse, etc.). An antibody that comprises at least one constant region of In some embodiments, a chimeric antibody comprises at least one murine variable region and at least one canine constant region. In some embodiments, a chimeric antibody comprises at least one murine variable region and at least one feline constant region. In some embodiments, all of the variable regions of the chimeric antibody are from a first species and all of the constant regions of the chimeric antibody are from a second species. In some embodiments, a chimeric antibody comprises a constant heavy chain region or a constant light chain region from a companion animal. In some embodiments, the chimeric antibody comprises murine variable heavy and light chains and companion animal constant heavy and light chains. For example, the chimeric antibody may comprise murine variable heavy and light chains and dog constant heavy and light chains, wherein the chimeric antibody comprises murine variable heavy and light chains and feline constant heavy and light chains. Alternatively, the chimeric antibody may comprise mouse variable heavy and light chains and horse constant heavy and light chains.

In some embodiments, the anti-IL31 antibody is
a. (i) the light chain amino acid sequence of SEQ ID NO:26, (ii) the heavy chain amino acid sequence of SEQ ID NO:27, or (iii) the light chain amino acid sequence of (i) and the heavy chain sequence of (ii), or b. (i) the light chain amino acid sequence of SEQ ID NO:30, (ii) the heavy chain amino acid sequence of SEQ ID NO:31, or (iii) the light chain amino acid sequence of (i) and the heavy chain sequence of (ii) include.

A "canine chimera" or "canine chimeric antibody" refers to a chimeric antibody having at least a portion of the heavy chain or a portion of the light chain derived from a dog. A "feline chimera" or "feline chimeric antibody" refers to a chimeric antibody having at least a portion of the heavy chain or a portion of the light chain derived from a cat. "Equine chimera" or "equine chimeric antibody" means a chimeric antibody having at least a portion of the heavy chain or a portion of the light chain derived from a horse. In some embodiments, a canine chimeric antibody comprises murine variable heavy and light chains and canine constant heavy and light chains. In some embodiments, a feline chimeric antibody comprises murine variable heavy and light chains and feline constant heavy and light chains. In some embodiments, the equine chimeric antibody comprises murine variable heavy and light chains and equine constant heavy and light chains. In some embodiments, the antibody is a chimeric antibody comprising a murine variable heavy chain framework region or a murine variable light chain framework region.

A "canine antibody," as used herein, is an antibody produced in a dog, an antibody produced in a non-canine animal that comprises canine immunoglobulin genes or comprises a canine immunoglobulin peptide, or an antibody repertoire It includes antibodies selected using in vitro methods such as phage display, which are based on immunoglobulin sequences. The term "canine antibody" refers to the genus of sequences that are canine sequences. Thus, the term does not refer to the process of creating an antibody, but rather to a genus of related sequences.

In some embodiments, the anti-IL31 antibody comprises a canine heavy chain constant region selected from IgG-A, IgG-B, IgG-C, and IgG-D constant regions. In some embodiments, the anti-IL31 antibody is a canine IgG-A, IgG-B, IgG-C, or IgG-D antibody. In some embodiments, the anti-IL31 antibody is (a) a canine IgG-A antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 17, (b) a canine IgG-B antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 18; (c) a canine IgG-C antibody comprising the heavy chain amino acid sequence of SEQ ID NO:19; or (d) a canine IgG-D antibody comprising the heavy chain amino acid sequence of SEQ ID NO:20.

A "feline antibody," as used herein, is an antibody produced in a cat, an antibody produced in a non-feline animal that comprises a feline immunoglobulin gene or comprises a feline immunoglobulin peptide, or an antibody repertoire It includes antibodies selected using in vitro methods such as phage display, which are based on immunoglobulin sequences. The term "feline antibody" refers to the genus of sequences that are feline sequences. Thus, the term does not refer to the process of creating an antibody, but rather to a genus of related sequences.

In some embodiments, the anti-IL31 antibody comprises a feline heavy chain constant region selected from IgG1, IgG2a, and IgG2b constant regions. In some embodiments, the anti-IL31 antibody is a feline IgG1, IgG2a, or IgG2b antibody.

An "equine antibody," as used herein, is an antibody produced in an equine animal, an antibody produced in a non-equine animal that contains equine immunoglobulin genes or contains equine immunoglobulin peptides, or an antibody repertoire It includes antibodies selected using in vitro methods such as phage display, which are based on immunoglobulin sequences. The term "equine antibody" refers to the genus of sequences that are equine sequences. Thus, the term does not refer to the process of creating an antibody, but rather to a genus of related sequences.

In some embodiments, the anti-IL31 antibody comprises an equine heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 constant regions. In some embodiments, the anti-IL31 antibodies are horse IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 antibodies.

A "canine antibody" refers to an antibody in which at least one amino acid in a portion of a non-canine variable region has been replaced with the corresponding amino acid from a canine variable region. In some embodiments, a canine antibody comprises at least one canine constant region (eg, gamma constant region, alpha constant region, delta constant region, epsilon constant region, mu constant region, or others) or a fragment thereof. In some embodiments, caninized antibodies are Fab, scFv, (Fab') ₂ , and other antibody fragments. The term "caninized" refers to a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof comprising the minimal sequence of a non-canine immunoglobulin (Fv, Fab, Fab', F(ab') ₂ , or other antigen-binding antibody sequence, etc.). Caninized antibodies are those in which residues from the CDRs of the recipient are replaced by residues from the CDRs of a non-canine species such as mouse, rat, or rabbit (donor antibody) with the desired specificity, affinity, and potency. canine immunoglobulin (recipient antibody). In some instances, Fv framework region (FR) residues of the canine immunoglobulin are replaced by corresponding non-canine residues. Furthermore, caninized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.

In some embodiments, at least one amino acid residue in a portion of the mouse variable heavy chain or mouse variable light chain is replaced with the corresponding amino acid from the canine variable region. In some embodiments, the modified chain is fused to a constant canine heavy chain or a constant canine light chain. In some embodiments, the anti-IL31 antibody comprises (a) the heavy chain sequence of SEQ ID NO: 15, (b) the heavy chain sequence of SEQ ID NO: 17, (c) the heavy chain sequence of SEQ ID NO: 18, (d) SEQ ID NO: 19, (e) the heavy chain sequence of SEQ ID NO:20, (f) the light chain sequence of SEQ ID NO:16, or (g) the light chain sequence of SEQ ID NO:21.

A "feline antibody" refers to an antibody in which at least one amino acid in a portion of the non-feline variable region has been replaced with the corresponding amino acid from the feline variable region. In some embodiments, a feline antibody comprises at least one feline constant region (eg, gamma constant region, alpha constant region, delta constant region, epsilon constant region, mu constant region, or others) or fragment thereof. In some embodiments, the feline antibody is a Fab, scFv, (Fab') ₂ or other antibody fragment. The term “felineized” refers to chimeric immunoglobulins, immunoglobulin chains, or fragments thereof that contain the minimal sequence of a non-feline immunoglobulin (Fv, Fab, Fab′, F(ab′) ₂ , or other antigen-binding antibody sequence, etc.). A feline antibody is one in which residues from the CDRs of the recipient are replaced by residues from the CDRs of a non-feline species such as mouse, rat, or rabbit (donor antibody) with the desired specificity, affinity, and potency. and feline immunoglobulin (recipient antibody). In some instances, Fv framework region (FR) residues of the feline immunoglobulin are replaced by corresponding non-feline residues. Furthermore, feline antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.

In some embodiments, at least one amino acid residue in a portion of the mouse variable heavy chain or mouse variable light chain is replaced with the corresponding amino acid from the feline variable region. In some embodiments, the modified chain is fused to a constant heavy cat chain or a constant canine light chain. In some embodiments, the anti-IL31 antibody comprises (a) the light chain sequence of SEQ ID NO:32, (b) the light chain sequence of SEQ ID NO:34, (c) the heavy chain sequence of SEQ ID NO:33, or (d) the sequence It is a feline antibody containing the heavy chain sequence numbered 35.

By "equine antibody" is meant an antibody in which at least one amino acid in a portion of the non-equine variable region has been replaced with the corresponding amino acid from the equine variable region. In some embodiments, an equine antibody comprises at least one equine constant region (eg, gamma constant region, alpha constant region, delta constant region, epsilon constant region, mu constant region, or others) or fragment thereof. In some embodiments, the equine antibody is a Fab, scFv, (Fab') ₂ , or other antibody fragment. The term "equine" refers to a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof containing the minimal sequence of a non-equine immunoglobulin (Fv, Fab, Fab', F(ab') ₂ , or other antigen-binding antibody sequence, etc.). Equineized antibodies are those in which residues from the CDRs of the recipient are replaced by residues from the CDRs of a non-equine species such as mouse, rat, or rabbit (donor antibody) with the desired specificity, affinity, and potency. Horse immunoglobulin (recipient antibody) may be included. In some instances, Fv framework region (FR) residues of the equine immunoglobulin are replaced by corresponding non-equine residues. Furthermore, equine antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.

In some embodiments, at least one amino acid residue in a portion of the mouse variable heavy chain or mouse variable light chain is replaced with the corresponding amino acid from the equine variable region. In some embodiments, the modified chain is fused to a horse constant heavy chain or a dog constant light chain.

The term "IgX Fc" means that the Fc region is derived from a particular antibody isotype (eg, IgG, IgA, IgD, IgE, IgM, etc.), where "X" refers to the antibody isotype. Thus, "IgG Fc" refers to the Fc region of the γ chain, "IgA Fc" refers to the Fc region of the α chain, "IgD Fc" refers to the Fc region of the δ chain, and "IgE Fc" refers to the ε chain. chain, "IgM Fc" means the Fc region of the μ chain, and so on. In some embodiments, the IgG Fc region comprises CH1, hinge, CH2, CH3, and CL1. "IgX-N-Fc" means that the Fc region belongs to a specific subclass of antibody isotype (e.g., canine IgG subclasses A, B, C, or D, feline IgG subclasses 1, 2a, or 2b, or equine IgG subclasses IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7, etc.), where "N" means subclass. In some embodiments, the IgX Fc or IgX-N-Fc region is from a companion animal such as a dog, cat, or horse. In some embodiments, the IgG Fc region is isolated from a canine gamma heavy chain such as IgG-A, IgG-B, IgG-C, or IgG-D. In some examples, the IgG Fc region is isolated from a feline gamma heavy chain such as IgG1, IgG2a, or IgG2b. Antibodies comprising an IgG-A, IgG-B, IgG-C, or IgG-D Fc region can provide high expression levels in recombinant production systems.

The term "affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (eg an antibody) and its binding partner (eg an antigen). The affinity of molecule X for its partner Y can generally be expressed as a dissociation constant (K _D ). Affinity can be measured by common methods known in the art, such as immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance devices.

The terms "K _D ", "K _d ", "Kd", or "Kd value" are used interchangeably and refer to the equilibrium dissociation constant of an antibody-antigen interaction. In some embodiments, the K _d of an antibody is measured using biolayer interferometry using a biosensor such as the Octet® system (Pall ForteBio LLC, Fremont, Calif.), according to the supplier's instructions. . Briefly, biotinylated antigen is bound to a sensor chip and antibody association is monitored for 90 seconds and dissociation for 600 seconds. The buffer for the dilution and binding steps is 20 mM phosphate, 150 mM NaCl, pH 7.2. A buffer-only blank curve is subtracted to correct for any drift. Data are fit to a 2:1 binding model using ForteBio data analysis software to determine association rate constants (k _on ), dissociation rate constants (k _off ), and K _d . The equilibrium dissociation constant (K _d ) is calculated as the ratio of k _off /k _on . The term "k _on " refers to the rate constant of antibody to antigen association, and the term "k _off " refers to the rate constant of antibody dissociation from an antibody/antigen complex.

The term "binding" to an antigen or epitope is a term well understood in the art and methods of determining such binding are also well known in the art. the molecule reacts with, associates with, or has an affinity for a particular cell or substance, and the reaction, association, or affinity is determined by one or more methods known in the art, such as immunoblotting; A molecule is said to exhibit "binding" if it is detectable by ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance devices, etc.

"Surface Plasmon Resonance" is a real-time two-dimensional measurement by detecting changes in protein concentration within a biosensor matrix using, for example, the BIAcore™ system (BIACore International AB, GE Healthcare Company, Uppsala, Sweden and Piscataway, N.J.). Represents an optical phenomenon that allows the analysis of bispecific interactions. Further description includes Jonsson et al. (1993) Ann. Biol. Clin. 51:19-26.

"Biolayer interferometry" refers to an optical analysis technique that analyzes the interference pattern of light reflected from a layer of protein immobilized on a biosensor chip and an internal reference layer. A change in the number of molecules bound to the biosensor chip changes the interference pattern, which can be measured in real time. A non-limiting exemplary device for biolayer interferometry is the Octet® System (Pall ForteBio LLC). For example, Abdiche et al., 2008, Anal. Biochem. 377:209-277.

In some embodiments, the anti-IL31 antibody is less than 5×10 ⁻⁶ M, less than 1×10 ⁻⁶ M, less than 5×10 ⁻⁷ M, 1×10 ⁻⁷ M as measured by biolayer interferometry less than 5×10 ⁻⁸ M, less than 1×10 ⁻⁸ M, less than 5×10 ⁻⁹ M, less than 1×10 ⁻⁹ M, less than 5×10 ⁻¹⁰ M, less than 1×10 ⁻¹⁰ M, to canine IL31, feline IL31, or equine IL31 with a dissociation constant (Kd) of less than 5×10 ⁻¹¹ M, less than 1×10 ⁻¹¹ M, less than 5× ^{10 −12 M} , or less than 1×10 −12 M Join. In some embodiments, the anti-IL31 antibody is 5×10 ⁻⁶ M to 1×10 −6 M, 5×10 −6 M to 5×10 ⁻⁷ M, 5×10 −6 M to 1×10 −6 M, 5×10 ⁻⁶ M to 1×10 −6 M, 5×10 −6 M to 5×10 ⁻⁷ M, as measured by biolayer interferometry. 10 ⁻⁶ M to 1×10 ⁻⁷ M, 5×10 ⁻⁶ M to 5×10 ⁻⁸ M, 5×10 ⁻⁶ M to 1×10 −8 M, 5× ^{10 −6 M} to 5×10 ⁻⁹ M, 5×10 ⁻⁶ M to 1×10 ⁻⁹ M, 5×10 ⁻⁶ M to 5×10 ⁻¹⁰ M, 5×10 ⁻⁶ M to 1×10 ⁻¹⁰ M, 5×10 ^{− 6} M to 5×10 ⁻¹¹ M, 5×10 ⁻⁶ M to 1×10 ⁻¹¹ M, 5×10 ⁻⁶ M to 5×10 ⁻¹² M, 5×10 ⁻⁶ M to 1×10 ⁻¹² M, 1×10 ⁻⁶ M to 5×10 ⁻⁷ M, 1×10 ⁻⁶ M to 1×10 ⁻⁷ M, 1×10 ⁻⁶ M to 5×10 ⁻⁸ M, 1×10 ⁻⁶ M to 1×10 ⁻⁸ M, 1×10 ⁻⁶ M to 5×10 ⁻⁹ M, 1×10 ⁻⁶ M to 1×10 ^{−9 M, 1×10 −6 M} to 5×10 ⁻¹⁰ M, 1×10 ⁻⁶ M to 1×10 ⁻¹⁰ M, 1×10 ⁻⁶ M to 5×10 ⁻¹¹ M, 1×10 ⁻⁶ M to 1×10 ⁻¹¹ M, 1×10 ⁻⁶ M to 5 ×10 ⁻¹² M, 1×10 ⁻⁶ M to 1×10 ⁻¹² M, 5×10 ⁻⁷ M to 1×10 −7 M, 5×10 ^{−7 M} to 5×10 ⁻⁸ M, 5× 10 ⁻⁷ M to 1×10 ⁻⁸ M, 5×10 ⁻⁷ M to 5×10 ⁻⁹ M, 5×10 ⁻⁷ M to 1×10 −9 M, 5× ^{10 −7 M} to 5×10 ⁻¹⁰ M, 5×10 ⁻⁷ M to 1×10 ⁻¹⁰ M, 5×10 ⁻⁷ M to 5×10 ⁻¹¹ M, 5×10 ⁻⁷ M to 1×10 ⁻¹¹ M, 5×10 ^{− 7} M to 5×10 ⁻¹² M, 5×10 ⁻⁷ M to 1×10 ⁻¹² M, 1×10 ⁻⁷ M to 5×10 ⁻⁸ M, 1×10 ⁻⁷ M to 1×10 ⁻⁸ M, 1×10 ⁻⁷ M to 5×10 ⁻⁹ M, 1×10 ⁻⁷ M to 1×10 ⁻⁹ M, 1×10 ⁻⁷ M to 5×10 ⁻¹⁰ M, 1×10 ⁻⁷ M to 1×10 ⁻¹⁰ M, 1×10 ⁻⁷ M to 5×10 ⁻¹¹ M, 1×10 ⁻⁷ M to 1×10 ^{−11 M, 1×10 −7 M} to 5×10 ⁻¹² M, 1×10 ⁻⁷ M to 1×10 ⁻¹² M, 5×10 ^{−8 M to 1×10 −8} M, 5×10 ⁻⁸ M to 5×10 ⁻⁹ M, 5× ^{10 −8 M} to 1 ×10 ⁻⁹ M, 5×10 ⁻⁸ M to 5×10 ⁻¹⁰ M, 5×10 ⁻⁸ M to 1×10 ⁻¹⁰ M, 5×10 ⁻⁸ M to 5×10 ⁻¹¹ M, 5× 10 ⁻⁸ M to 1×10 ⁻¹¹ M, 5×10 ⁻⁸ M to 5×10 ⁻¹² M, 5×10 ⁻⁸ M to 1×10 ^{−12 M, 1×10 −8 M} to 5×10 ⁻⁹ M, 1×10 ⁻⁸ M to 1×10 ⁻⁹ M, 1×10 ⁻⁸ M to 5×10 ⁻¹⁰ M, 1×10 ⁻⁸ M to 1×10 ⁻¹⁰ M, 1×10 ^{− 8} M to 5×10 ⁻¹¹ M, 1×10 ⁻⁸ M to 1×10 ⁻¹¹ M, 1×10 ⁻⁸ M to 5×10 ⁻¹² M, 1×10 ⁻⁸ M to 1×10 ⁻¹² M, 5×10 ⁻⁹ M to 1×10 ⁻⁹ M, 5×10 ⁻⁹ M to 5×10 ⁻¹⁰ M, 5×10 ⁻⁹ M to 1×10 ⁻¹⁰ M, 5×10 ⁻⁹ M to 5×10 ⁻¹¹ M, 5×10 ⁻⁹ M to 1×10 ⁻¹¹ M, 5×10 ⁻⁹ M to 5×10 ⁻¹² M, 5×10 ⁻⁹ M to 1×10 ⁻¹² M, 1×10 ⁻⁹ M to 5×10 ⁻¹⁰ M, 1×10 ⁻⁹ M to 1×10 ⁻¹⁰ M, 1×10 ⁻⁹ M to 5×10 ⁻¹¹ M, 1×10 ⁻⁹ M to 1 ×10 ⁻¹¹ M, 1×10 ⁻⁹ M to 5×10 ⁻¹² M, 1×10 ⁻⁹ M to 1×10 ⁻¹² M, 5×10 ⁻¹⁰ M to 1×10 ⁻¹⁰ M, 5× 10 ⁻¹⁰ M to 5×10 ⁻¹¹ M, 1×10 ⁻¹⁰ M to 5×10 ⁻¹¹ M, 1×10 ⁻¹⁰ M to 1×10 ⁻¹¹ M, 1×10 ⁻¹⁰ M to 5×10 ⁻¹² M, 1×10 ⁻¹⁰ M to 1×10 ⁻¹² M, 5×10 ⁻¹¹ M to 1×10 ⁻¹² M, 5×10 ⁻¹¹ M to 5×10 ⁻¹² M, 5×10 ^{− Canine IL31 , feline} IL31 ^, or ^horse Binds to IL31. In some embodiments, the anti-IL31 antibody binds to canine IL31, feline IL31, or equine IL31 as determined by immunoblot analysis.

In some embodiments, the anti-IL31 antibody does not bind human IL31 as determined by immunoblot analysis and/or biolayer interferometry.

In some embodiments, anti-IL31 antibodies are provided that compete with an anti-IL31 antibody described herein (eg, M14, M18, M19, or M87) for binding to IL31. In some embodiments, antibodies can be made or used that compete for binding with any of the antibodies provided herein. In some embodiments, an anti-IL31 antibody is provided that competes with a monoclonal M14 antibody for binding canine IL31 or feline IL31.

A "variant" is one that after aligning the sequences and introducing gaps where necessary to maximize the percent sequence identity, without considering any conservative substitutions as part of the sequence identity. A biologically active polypeptide having at least about 50% amino acid sequence identity with the native sequence polypeptide is meant. Such variants include, for example, polypeptides with one or more amino acid residues added or deleted at the N-terminus or C-terminus of the polypeptide.

In some embodiments, a variant has at least about 50% amino acid sequence identity, at least about 60% amino acid sequence identity, at least about 65% amino acid sequence identity, at least about 70% amino acid sequence identity, or at least about 70% amino acid sequence identity with the native sequence polypeptide. at least about 75% amino acid sequence identity, at least about 80% amino acid sequence identity, at least about 85% amino acid sequence identity, at least about 90% amino acid sequence identity, at least about 95% amino acid sequence identity.

As used herein, "% amino acid sequence identity" and "homology" with respect to peptide, polypeptide, or antibody sequences are used to align the sequences and maximize the percent sequence identity. Amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, without regard to any conservative substitutions as part of the sequence identity, after introducing gaps where necessary. Defined as a percentage of the group. Alignment for purposes of determining percent amino acid sequence identity may be performed using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINE™ (DNASTAR) software, within the skill of the art. It can be achieved by various methods. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

Amino acid substitutions can include, but are not limited to, replacement of one amino acid with another amino acid in a polypeptide. Exemplary substitutions are shown in Table 2. Amino acid substitutions may be introduced into the antibody of interest and the products screened for the desired activity, eg, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
Table 2

Amino acids can be classified according to common side chain properties.
(1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that affect chain orientation: Gly, Pro;
(6) Aromatics: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

In some embodiments, the anti-IL31 antibody comprises a heavy chain and a light chain,
a. the heavy chain has a CDR-H1 sequence that has at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:1; at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:62, SEQ ID NO:89, or SEQ ID NO:87 a CDR-H2 sequence having identity and having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:3; comprising a CDR-H3 sequence having
b. A light chain has a CDR that has at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:63 - a CDR-L2 sequence that has at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the L1 sequence, the amino acid sequence of SEQ ID NO:9; and CDR-L3 sequences having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:10.

In some embodiments, the anti-IL31 antibody comprises a heavy chain and a light chain,
a. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:24; (ii) at least 85% with the amino acid sequence of SEQ ID NO:25; , a variable heavy chain sequence having at least 90%, at least 95%, or at least 98% sequence identity, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or b. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:16; (ii) at least 85% with the amino acid sequence of SEQ ID NO:15; , a variable heavy chain sequence having at least 90%, at least 95%, or at least 98% sequence identity, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or c. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:32; (ii) at least 85% with the amino acid sequence of SEQ ID NO:33; , a variable heavy chain sequence having at least 90%, at least 95%, or at least 98% sequence identity, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or d. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:64; (ii) at least 85% with the amino acid sequence of SEQ ID NO:67; , a variable heavy chain sequence having at least 90%, at least 95%, or at least 98% sequence identity, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or e. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:65; (ii) at least 85% with the amino acid sequence of SEQ ID NO:68; , a variable heavy chain sequence having at least 90%, at least 95%, or at least 98% sequence identity, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or f. (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:66; (ii) at least 85% with the amino acid sequence of SEQ ID NO:69; , a variable heavy chain sequence having at least 90%, at least 95%, or at least 98% sequence identity, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii).

The term "vector" is used to describe a polynucleotide that can be cloned or engineered to contain a polynucleotide that can be propagated in a host cell. A vector may comprise the following elements: an origin of replication, one or more regulatory sequences (such as promoters or enhancers) that regulate the expression of the polypeptide of interest, or one or more selectable marker genes (such as an antibiotic resistance gene). and genes that can be used in colorimetric assays, such as β-galactosidase). The term "expression vector" means a vector that is used to express a polypeptide of interest in a host cell.

By "host cell" is meant a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate cells, fungal cells such as yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NS0 cells, PER. C6® cells (Crucell), 293 cells, and CHO cells, and derivatives thereof such as 293-6E, DG44, CHO-S, and CHO-K cells. A host cell includes the progeny of a single host cell, which progeny are not necessarily completely (in morphological or genomic DNA complementarity) with the original parent cell, due to natural, inadvertent, or deliberate mutations. They do not have to be identical. A host cell includes cells transfected in vivo with a polynucleotide encoding an amino acid sequence provided herein.

The term "isolated," as used herein, means a molecule that is separated from at least some of the components with which it is typically found or produced in nature. For example, a polypeptide is said to be "isolated" when it is separated from at least some of the components of the cell that produced it. If the polypeptide is secreted from the cell following expression, physical separation of the supernatant containing the polypeptide from the cells that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide may be referred to as a polynucleotide unless it is part of a larger polynucleotide (such as genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) in which it is typically found in nature; A nucleotide is said to be "isolated" if it is separated from at least some of the components of the cell that produced it. Thus, a DNA polynucleotide contained in a vector within a host cell may be referred to as "isolated." In some embodiments, anti-IL31 antibodies are purified using chromatography, such as size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.

The term "companion animal species" means an animal suitable to be a companion to humans. In some embodiments, the companion animal species is a small mammal, such as a canine, feline, dog, cat, horse, rabbit, ferret, guinea pig, rodent, and the like. In some embodiments, the companion animal species is horses, cows, pigs, and other livestock.

The term "IL31 signaling function" refers to any one or combination of downstream activities that occur upon binding of IL31 to its receptor or receptor complex. In some embodiments, the IL31 signaling function comprises activation of Janus kinase (Jak) 1 or Jak2 signaling molecules. In some embodiments, the IL31 signaling function comprises phosphorylation of STAT-3 or STAT-5 proteins. In some embodiments, the IL31 signaling function comprises activating the ERK1/2 MAP kinase signaling pathway. In some embodiments, the IL31 signaling function comprises activating the PI3K/AKT signaling pathway. In some embodiments, the IL31 signaling function comprises activating the Jak1/2 signaling pathway.

"STAT phosphorylation" means alteration by phosphorylation after expression of a STAT protein. For example, "STAT-3 phosphorylation" means phosphorylation of STAT-3 and "STAT-5 phosphorylation" means phosphorylation of STAT-5. In some embodiments, STAT-3 phosphorylation is measured by immunoblot analysis. For example, cells (e.g., canine monocyte DH82 cells) were treated with 15% heat-inactivated fetal bovine serum, 2 mmol/L GlutaMax, 1 mmol/L sodium pyruvate, and 10 nm/mL canine interferon-c (R&D Systems, Minneapolis, Minn., USA) containing anti-IL31 as described herein at a density of 1×10 ⁵ cells/well in a growth medium (e.g. MEM, Life Technologies®) in 96-well cell culture plates. Inoculate for 24 hours at 37° C. in the presence of antibody. Immunoblot analysis of cell lysates using anti-phospho-STAT-3 and anti-STAT-3 antibodies (R&D Systems) was used to detect the concentrations of phosphorylated and non-phosphorylated STAT-3 relative to each other. , compared to the β-actin control. Methods for determining qualitative or quantitative protein concentration by immunoblot are understood by those of skill in the art. In some embodiments, relative concentrations are determined qualitatively by visual inspection of immunoblots. In some embodiments, concentrations of phosphorylated STAT-3 and non-phosphorylated STAT-3 are determined by digitally imaging the immunoblot, determining the intensity of the bands, and creating a linear standard line of known concentrations of STAT-3 protein. is determined quantitatively by back-calculating the concentration of phosphorylated or non-phosphorylated STAT-3 in the sample using

"Reduce" or "inhibit" means to decrease, reduce or stop an activity, function or amount relative to a reference. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 20% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more. In some embodiments, the amount is inhibited or reduced over a period of time compared to a control dose (such as placebo) over the same period. "Reference," as used herein, means any sample, standard, or level used for comparison purposes. References may be obtained from healthy or non-diseased samples. In some examples, the reference is obtained from a non-diseased or non-treated sample of the companion animal. In some examples, the reference is obtained from one or more healthy animals of a particular species other than the animal being tested or treated.

The term "substantially reduced", as used herein, means that the difference between two values is statistically significant within the context of the biological property measured by the numerical value and the reference numerical value. It represents the degree of reduction between the numerical value and the reference numerical value that is high enough for the trader to consider. In some embodiments, the substantially reduced numerical value is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60% compared to the reference value. %, 70%, 80%, 90%, or 100% reduction.

In some embodiments, the IL31 antibody reduces IL31 signaling function in the absence of the antibody by at least 10%, at least 15%, at least 20%, at least as measured by reduction in STAT-3 phosphorylation. 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% IL31 signaling in a companion animal species function may be reduced. In some embodiments, the reduction in IL31 signaling function or the reduction in STAT-3 phosphorylation is 10%-15%, 10%-20%, 10%-25%, 10%-30%, 10%- 35%, 10%-40%, 10%-45%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%, 10%-100% , 15%-20%, 15%-25%, 15%-30%, 15%-35%, 15%-40%, 15%-45%, 15%-50%, 15%-60%, 15 %~70%, 15%~80%, 15%~90%, 15%~100%, 20%~25%, 20%~30%, 20%~35%, 20%~40%, 20%~ 45%, 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20%-100%, 25%-30%, 25%-35% , 25%-40%, 25%-45%, 25%-50%, 25%-60%, 25%-70%, 25%-80%, 25%-90%, 25%-100%, 30 % ~ 35%, 30% ~ 40%, 30% ~ 45%, 30% ~ 50%, 30% ~ 60%, 30% ~ 70%, 30% ~ 80%, 30% ~ 90%, 30% ~ 100%, 35%-40%, 35%-45%, 35%-50%, 35%-60%, 35%-70%, 35%-80%, 35%-90%, 35%-100% , 40%-45%, 40%-50%, 40%-60%, 40%-70%, 40%-80%, 40%-90%, 40%-100%, 45%-50%, 45 % ~ 60%, 45% ~ 70%, 45% ~ 80%, 45% ~ 90%, 45% ~ 100%, 50% ~ 60%, 50% ~ 70%, 50% ~ 80%, 50% ~ 90%, 50%-100%, 60%-70%, 60%-80%, 60%-90%, 60%-100%, 70%-80%, 70%-90%, 70%-100% , 80%-90%, 80%-100%, or 90%-100%.

Pharmaceutical Compositions The terms "pharmaceutical formulation" and "pharmaceutical composition" mean that the active ingredient is in a form that allows the biological activity of the active ingredients to be effective and is unacceptable to the subject to whom the formulation is to be administered. means a preparation that does not contain additional ingredients that are as toxic as

"Pharmaceutically acceptable carrier" means any conventional non-toxic solid, semi-solid, or liquid filler in the art for use with therapeutic agents, together with a "pharmaceutical composition" for administration to a subject. , diluent, encapsulating material, formulation aid, or carrier. A pharmaceutically acceptable carrier is nontoxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. Pharmaceutically acceptable carriers are preferred for the formulations employed. Examples of pharmaceutically acceptable carriers include alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, canine or other animal albumin, phosphate, citrate, tromethamine, HEPES buffers, and the like. buffers of glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, or magnesium tri Salts or electrolytes such as silicates, polyvinylpyrrolidone, cellulosics, polyethylene glycol, sucrose, mannitol, or amino acids including, but not limited to, arginine.

Pharmaceutical compositions can be stored in lyophilized form. Accordingly, in some embodiments, the preparation process includes a lyophilization step. The lyophilized composition can then be reformulated, typically as an aqueous composition suitable for parenteral administration, prior to administration to a dog, cat, or horse. In other embodiments, the pharmaceutical composition is administered to dogs, cats, or horses as a liquid, either directly or with appropriate dilution, especially when the antibody is highly stable to denaturation by heat and oxidation. It can be stored as an aqueous composition that can be administered. Lyophilized compositions can be reconstituted with sterile water for injection (WFI). Antimicrobial agents (eg, bacteriostatic agents such as benzyl alcohol) may be included. Thus, the present invention provides pharmaceutical compositions in solid or liquid form.

The pH of the pharmaceutical composition may range from about pH 5 to about pH 8 when administered. The compositions of the invention are sterile when used for therapeutic purposes. Sterility can be achieved by any of several means known in the art including filtration through sterile filtration membranes (eg, 0.2 micron membranes). Sterility may be maintained in the presence or absence of antimicrobial agents.

In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition has a pH of 5.0-6.2, 5.0-6.0, or 5.3-5.7. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5 .7, 5.8, 5.9, 6.0, 6.1, or 6.2.

In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition comprises L-histidine, sodium chloride, and polysorbate 80.

In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition is 80 nM-200 nM, 100 nM-180 nM, 100 nM-175 nM, 110 nM-170 nM, 120 nM-160 nM, 120 nM-150 nM, 130 nM-150 nM, 130 nM Contains sodium chloride at a concentration of ˜160 nM, 100 nM, 80 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, or 200 nM.

In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition is 0.005 mg/mL to 0.5 mg/mL, 0.01 mg/mL to 0.1 mg/mL, 0.1 mg/mL ~0.5 mg/mL, 0.005 mg/mL to 0.01 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.05 mg/mL, 0 Polysorbate 80 at a concentration of 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL, or 0.1 mg/mL.

In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition is 5 mM to 100 mM, 10 mM to 50 mM, 20 mM to 30 mM, 10 mM to 30 mM, 20 mM to 80 mM, 30 mM to 70 mM, 40 mM to 60 mM, 10 mM , 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM L-histidine.

In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition comprises m-cresol or benzyl alcohol. In some embodiments, the concentration of m-cresol is about 0.2%, about 0.1% to about 0.3%, about 0.08% to about 0.25%, or about 0.05% to about 0.25%. In some embodiments, the concentration of benzyl alcohol is about 1%, about 0.5% to about 2%, about 0.2% to about 2.5%, about 1% to about 5%, about 0.5% % to about 5%, or about 1% to about 3%.

In some embodiments the pharmaceutically acceptable carrier or pharmaceutical composition comprises a sugar. In some embodiments, the sugar is sucrose, trehalose, D-mannitol, maltose, and/or sorbitol. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition is 0.5%-20%, 1%-10%, 1%-5%, 1%-3%, 0.5% %, 1%, 2%, 3%, 4%, 5%, or 10% sugar.

In some embodiments, a pharmaceutically acceptable carrier or pharmaceutical composition comprises an antimicrobial agent. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition comprises m-cresol or methylparaben. In some embodiments, the pharmaceutically acceptable carrier or pharmaceutical composition comprises 0.2% m-cresol. In some embodiments the pharmaceutically acceptable carrier or pharmaceutical composition comprises 0.9% methylparaben.

Uses of Antibodies and Pharmaceutical Compositions Antibodies of the invention or pharmaceutical compositions comprising antibodies may be useful for treating IL31-induced conditions. As used herein, "IL31-induced condition" means a disease associated with, caused by, or characterized by elevated levels or altered slopes of IL31 concentration. Such IL31-induced conditions include, but are not limited to, pruritic or allergic disorders. In some embodiments, the IL31-induced condition is atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, or eczema. An IL31-induced condition may be manifested in companion animals including, but not limited to, dogs, cats, or horses.

As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. "Treatment," as used herein, includes any administration or application of therapy for disease in a mammal, including a companion animal. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, reduction of disease severity, prevention or delay of disease spread, prevention of disease recurrence or slowing, slowing or slowing the progression of a disease, ameliorating the condition, arresting the disease or progression of the disease, arresting or slowing the disease or its progression, halting its progression, and remission (partial or complete) any one or more of Reducing the pathological consequences of proliferative disorders is also included in "treatment". The methods provided herein contemplate any one or more of these aspects of treatment. Consistent with the above, the term treatment does not require 100% elimination of all aspects of the disorder.

In some embodiments, anti-IL31 antibodies or pharmaceutical compositions comprising the same can be utilized in accordance with the methods herein for treating IL31-induced conditions. In some embodiments, an anti-IL31 antibody or pharmaceutical composition is administered to a companion animal such as a dog, cat, or horse to treat an IL31-induced condition.

A “therapeutically effective amount” of a substance/molecule, agonist or antagonist includes the type of disease to be treated, the condition, the severity and course of the disease, the type of treatment intended, previous therapy, if any, medical history, prior therapy It may vary according to factors such as response to stress, the discretion of the attending veterinarian, the age, sex, and weight of the animal, the ability of the substance/molecule, agonist, or antagonist to elicit the desired response from the animal. A therapeutically effective amount is also one in which any toxic or detrimental effects of a substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount means an amount effective, at dosages and for periods necessary, to achieve the desired therapeutic or prophylactic result.

In some embodiments, the anti-IL31 antibody or pharmaceutical composition comprising an anti-IL31 antibody is administered parenterally, subcutaneously, intravenously, or intramuscularly. In some embodiments, an anti-IL31 antibody or pharmaceutical composition comprising an anti-IL31 antibody is administered as a bolus injection or as a continuous infusion over a period of time. In some embodiments, the anti-IL31 antibody or pharmaceutical composition comprising an anti-IL31 antibody is administered intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intraarterially, intrasynovially, intrathecally, or by an inhalation route. be done.

The anti-IL31 antibodies described herein may be administered in amounts ranging from 0.1 mg/kg body weight to 100 mg/kg body weight per dose. In some embodiments, anti-IL31 antibodies may be administered in amounts ranging from 0.5 mg/kg body weight to 50 mg/kg body weight per dose. In some embodiments, anti-IL31 antibodies may be administered in amounts ranging from 1 mg/kg body weight to 10 mg/kg body weight per dose. In some embodiments, the anti-IL31 antibody ranges from 0.5 mg/kg body weight to 100 mg/kg body weight, from 1 mg/kg body weight to 100 mg/kg body weight, from 5 mg/kg body weight to 100 mg/kg body weight, 10 mg/kg body weight to 100 mg/kg body weight, 20 mg/kg body weight to 100 mg/kg body weight, 50 mg/kg body weight to 100 mg/kg body weight, 1 mg/kg body weight to 10 mg/kg body weight, 5 mg/kg body weight Amounts in the range of kg body weight to 10 mg/kg body weight, 0.5 mg/kg body weight to 10 mg/kg body weight, or 5 mg/kg body weight to 50 mg/kg body weight may be administered.

An anti-IL31 antibody or pharmaceutical composition comprising an anti-IL31 antibody can be administered to a companion animal at a single time or over a series of treatments. For example, an anti-IL31 antibody or a pharmaceutical composition comprising an anti-IL31 antibody may be administered at least one time, two or more times, at least two times, at least three times, at least four times, or at least five times.

In some embodiments, the dose is administered once weekly for at least two or three consecutive weeks, and in some embodiments, the treatment cycle is optionally separated by one or more treatment-free weeks. , is repeated two or more times. In other embodiments, the therapeutically effective dose is administered once daily for 2 to 5 consecutive days; in some embodiments, the treatment cycle is optionally followed by one or more treatment-free weeks. , and repeated twice or more.

Administration “in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) administration and consecutive or sequential administration in any order. The term "concurrently" is used herein to refer to two therapeutic agents in which at least a portion of the administration overlaps in time, or administration of one therapeutic agent occurs within a short period of time relative to administration of the other therapeutic agent. It is used to mean administration of the above therapeutic agents. For example, two or more therapeutic agents are administered temporally separated by about a specified few minutes or less. The term "sequentially" as used herein means that administration of one or more agents continues after administration of one or more other agents has been discontinued, or administration of one or more agents continues after administration of one or more other agents. is used to refer to the administration of two or more therapeutic agents when beginning prior to the administration of the agents. For example, administration of two or more therapeutic agents is administered at intervals of more than about a specified number of minutes. As used herein, "in combination with" means administration of one treatment modality in addition to another treatment modality. Thus, "in combination with" means administration of one treatment modality before, during, or after administration of another treatment modality to an animal.

In some embodiments, the method comprises administering a Jak inhibitor, PI3K inhibitor, AKT inhibitor, or MAPK inhibitor in combination with an anti-IL31 antibody or a pharmaceutical composition comprising an anti-IL31 antibody. . In some embodiments, the method provides an anti-IL17 antibody, an anti-TNFα antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD25 antibody, an anti-IL4 antibody in combination with an anti-IL31 antibody or a pharmaceutical composition comprising an anti-IL31 antibody. administering an antibody, an anti-IL13 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CD11α antibody, an anti-IL6R antibody, an anti-α4-integrin antibody, an anti-IL12 antibody, an anti-IL1β antibody, or an anti-BlyS antibody.

Provided herein are methods of exposing a cell to an anti-IL31 antibody or a pharmaceutical composition comprising an anti-IL31 antibody under conditions that allow binding of the antibody to IL31. In some embodiments, the cells are exposed to the antibody or pharmaceutical composition ex vivo. In some embodiments, cells are exposed to the antibody or pharmaceutical composition in vivo. In some embodiments, the cell is exposed to the anti-IL31 antibody or pharmaceutical composition under conditions that allow binding of the antibody to intracellular IL31. In some embodiments, cells are exposed to an anti-IL31 antibody or pharmaceutical composition under conditions that allow binding of the antibody to extracellular IL31. In some embodiments, the cells are administered in vivo by any one or more of the methods of administration described herein including, but not limited to, intraperitoneal, intramuscular, intravenous injection into a subject. may be exposed to the pharmaceutical composition. In some embodiments, cells may be exposed to an anti-IL31 antibody or pharmaceutical composition ex vivo by exposing the cells to media containing the antibody or pharmaceutical composition. In some embodiments, cell membrane permeability is achieved by any number of methods understood by those skilled in the art, such as subjecting the cells to electroporation prior to exposing the cells to media containing the antibody or pharmaceutical composition. or exposing the cells to a solution containing calcium chloride).

In some embodiments, binding reduces IL31 signaling function of the cell. In some embodiments, the IL31 antibody reduces STAT-3 phosphorylation by at least 10%, at least 15%, at least 20%, at least 25% compared to IL31 signaling function in the absence of the antibody, as measured by reduction in STAT-3 phosphorylation. %, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of IL31 signaling function in cells can be reduced. In some embodiments, the reduction in IL31 signaling function or the reduction in STAT-3 phosphorylation is 10%-15%, 10%-20%, 10%-25%, 10%-30%, 10%- 35%, 10%-40%, 10%-45%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%, 10%-100% , 15%-20%, 15%-25%, 15%-30%, 15%-35%, 15%-40%, 15%-45%, 15%-50%, 15%-60%, 15 %~70%, 15%~80%, 15%~90%, 15%~100%, 20%~25%, 20%~30%, 20%~35%, 20%~40%, 20%~ 45%, 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20%-100%, 25%-30%, 25%-35% , 25%-40%, 25%-45%, 25%-50%, 25%-60%, 25%-70%, 25%-80%, 25%-90%, 25%-100%, 30 % ~ 35%, 30% ~ 40%, 30% ~ 45%, 30% ~ 50%, 30% ~ 60%, 30% ~ 70%, 30% ~ 80%, 30% ~ 90%, 30% ~ 100%, 35%-40%, 35%-45%, 35%-50%, 35%-60%, 35%-70%, 35%-80%, 35%-90%, 35%-100% , 40%-45%, 40%-50%, 40%-60%, 40%-70%, 40%-80%, 40%-90%, 40%-100%, 45%-50%, 45 % ~ 60%, 45% ~ 70%, 45% ~ 80%, 45% ~ 90%, 45% ~ 100%, 50% ~ 60%, 50% ~ 70%, 50% ~ 80%, 50% ~ 90%, 50%-100%, 60%-70%, 60%-80%, 60%-90%, 60%-100%, 70%-80%, 70%-90%, 70%-100% , 80%-90%, 80%-100%, or 90%-100%.

Provided herein are methods of using anti-IL31 antibodies, polypeptides, and polynucleotides for the detection, diagnosis, and monitoring of IL31-induced conditions. Provided herein are methods of determining whether a companion animal will respond to anti-IL31 antibody treatment. In some embodiments, the method includes detecting whether the animal has cells expressing IL31 using an anti-IL31 antibody. In some embodiments, the detection method comprises contacting the sample with an antibody, polypeptide, or polynucleotide and determining whether the extent of binding differs from a reference or comparison sample (e.g., control). include. In some embodiments, the methods can be useful for determining whether an antibody or polypeptide described herein is an appropriate treatment for a subject animal.

In some embodiments the sample is a biological sample. The term "biological sample" means a quantity of a substance from a living organism or formerly living organism. In some embodiments, the biological sample is a cell or cell/tissue lysate. In some embodiments, biological samples include, but are not limited to, blood (eg, whole blood), plasma, serum, urine, synovial fluid, and epithelial cells.

In some embodiments, a cell or cell/tissue lysate is contacted with an anti-IL31 antibody and binding between the antibody and the cell is determined. A test cell that exhibits binding activity compared to a reference cell of the same tissue type indicates that the subject would benefit from treatment with an anti-IL31 antibody. In some embodiments, the test cells are from a companion animal tissue.

Various methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays that may be performed include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), turbidimetric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay ( RIA). Indicator moieties, or label groups, are capable of being attached to the antibody of interest and are selected to meet the needs of use in a variety of methods, often determined by the availability of assay equipment and compatible immunoassay procedures. be. Suitable labels include, but are not limited to, radionuclides (such as ¹²⁵ I, ¹³¹ I, ³⁵ S, ³ H, or ³² P), enzymes (such as alkaline phosphatase, horseradish peroxidase, luciferase, or p-glyctoate). sidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot™ nanoparticles supplied by Quantum Dot Corporation, Palo Alto, Calif.). included. General techniques used in performing the various immunoassays described above are known to those of skill in the art.

For diagnostic purposes, polypeptides, including antibodies, can be labeled with detectable moieties including, but not limited to, radioisotopes, fluorescent labels, and various enzyme-substrate labels known in the art. . Methods for conjugating labels to antibodies are known in the art. In some embodiments, the anti-IL31 antibody need not be labeled and its presence can be detected using a second labeled antibody that binds to the first anti-IL31 antibody. In some embodiments, anti-IL31 antibodies can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987). Anti-IL31 antibodies and polypeptides can also be used for in vivo diagnostic assays, such as in vivo imaging. Generally, antibodies or polypeptides are labeled with radionuclides (e.g., ¹¹¹ In, ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ¹²⁵ I, ³ H so that cells or tissues of interest can be localized using immunoscintiography). , or any other radionuclide label, including those outlined herein). Antibodies are also used as stains in pathology using techniques well known in the art.

In some embodiments, the first antibody is used diagnostically and the second antibody is used therapeutically. In some embodiments, the first antibody and the second antibody are different. In some embodiments, the first antibody and the second antibody can both bind to the antigen at the same time by binding to separate epitopes.

The following examples illustrate certain aspects of the disclosure and are not intended to limit the disclosure in any manner.

Example 1 Identification of Mouse Monoclonal Antibodies that Bind to Canine IL31 The canine IL31 gene encoding the IL31 protein (SEQ ID NO:22) was synthesized with a C-terminal poly-His tag and cloned into a mammalian expression vector. A plasmid carrying the canine IL31 gene was transfected into 293 cells.

The supernatant containing canine IL31 protein was collected and filtered. Canine IL31 was affinity purified using a Ni-NTA column (CaptivA® Protein A Affinity Resin, Repligen).

Mouse monoclonal antibodies were identified using standard immunizations with canine IL31 produced by 293 cells as the immunogen. Monoclonal antibodies were obtained by standard hybridoma technology using two different adjuvants during immunization (Antibody Solutions, Sunnyvale, Calif.). An enzyme-linked immunosorbent assay (ELISA) was developed to screen for clones producing IL31 binding antibodies. Canine IL31 was first biotinylated and then introduced into streptavidin-coated wells. Immunized sera were then added to the wells, washed and detected with HRP-conjugated anti-mouse antibody. A positive signal was generated by the presence of canine IL31 binding antibodies. Of the thousands of clones tested by ELISA, 170 clones with the highest binding affinity were selected based on signal intensity and further tested by biosensor assay (Forte Bio Octet). Biotinylated canine IL31 was bound to a sensor chip and supernatants of hybridoma clones containing anti-canine IL31 antibodies were selected based on the slow-off rate (rate of dissociation of antibody and ligand). The binding affinities of the top 19 candidates were measured at a single concentration and reported as equilibrium dissociation constants (Kd) after antibody concentration was determined by protein A titration assay using a Biosensor Octet. Each of the top 19 candidates had a Kd of less than 10 nM.

Additionally, each of the 170 clones with high binding activity based on ELISA were tested for neutralizing activity. To assess the activity of top candidates in reducing canine IL31-mediated pSTAT signaling using canine DH82 cells, the cell-based functional assay described below in Example 4 was performed. The top four clones (M14, M18, M19 and M87) were selected for further study. Notably, the majority of high affinity binders identified by ELISA were not functional.

Example 2: Identification of DNA sequences encoding VH and VL of monoclonal antibodies Hybridoma cells producing M14, M18, M19 and M87 were pelleted. RNA was extracted and cDNA was obtained using standard techniques using oligonucleotide primers to amplify mouse immunoglobulin (Ig) variable domains. The variable heavy (VH) and variable light (VL) chains of each of the four clones were sequenced and analyzed by sequence alignment (Figure 1, SEQ ID NOs:36-43). In particular, three of the four active antibodies (M14, M18, and M19) have an isoleucine at position 14 in the CDR-L1 of M18 (SEQ ID NO: 63), while M14 and M19 have a methionine at position 14. (SEQ ID NO: 8), and CDR-H2 of M18 has a tyrosine at position 9 (SEQ ID NO: 62 and SEQ ID NO: 87), while M14 and M19 have an aspartic acid at position 14 (SEQ ID NO: 2 and SEQ ID NO: 89 ), they share the same six CDR sequences. The CDR sequence similarities suggest that M14, M18 and M19 share common epitopes.

Example 3: Expression and Purification of Mouse-Canine Chimeric and Caninized IL31 mAb M14 from CHO Cells Mouse M14 VH (SEQ ID NO:25) and mouse VL (SEQ ID NO:24) to dog constant heavy chain and dog constant light chain A DNA sequence encoding a chimeric antibody was designed for the fusion. Nucleotide sequences were chemically synthesized and inserted into an expression vector suitable for transfection into CHO host cells. After transfection into CHO cells, light or heavy chain proteins or both were secreted from the cells. For example, chimeric M14 with canine IgG-B was purified by single step Protein A column chromatography.

Mouse M14 VH and VL were caninized by searching and selecting the correct canine germline antibody sequences as templates for CDR grafting and performing protein modeling. The caninized M14 IgG-B (SEQ ID NO: 18 and SEQ ID NO: 21) is readily expressed and subjected to protein A column or other chromatographic methods such as ion exchange column chromatography, hydrophobic interaction column chromatography, CHT, etc. Purification was in a single step by mixed mode column chromatography or multimodal mode column chromatography such as CaptoMMC. Low pH or other virus inactivation and virus removal steps can be applied. The purified protein is mixed with excipients and filter sterilized to prepare a pharmaceutical composition of the invention. A pharmaceutical composition is administered to a dog with atopic dermatitis in an amount sufficient to bind and inhibit IL31.

The vector was then used to perform pilot-scale transfections in CHO-S cells using FreestyleMax™ transfection reagent (Life Technologies). The supernatant was harvested by clarifying the conditioned medium. The protein was purified by a single pass Protein A chromatography step and used for further studies.

Example 4 Demonstration of IL31 Binding Activity This example demonstrates that antibodies of the invention represented by chimeric M14 (SEQ ID NO:26 and SEQ ID NO:27) and caninized M14 (SEQ ID NO:18 and SEQ ID NO:21) exhibit therapeutic activity. demonstrate binding to canine IL31 with kinetics requisite for

Binding analysis was performed using the biosensor Octet as follows. Briefly, canine IL31 was isolated at primary amine groups using EZ-Link™ NHS-LC-Biotin (ThermoScientific, Catalog No. 21336) or with EZ-Link™ Biotin-LC-Hydrazide (ThermoFisher Scientific). , Catalog No. 21340) were biotinylated at the glycan groups according to the manufacturer's instructions. Free, unreacted biotin was removed from biotinylated IL31 by extensive dialysis. Biotinylated canine IL31 was captured on a streptavidin sensor chip. Four different concentrations (400, 200, 66.6, and 33 nM) of chimeric and caninized M14 antibody association with human and canine IL31 (amine-conjugate-biotin), and 100 nM caninized M14 antibody Association with canine IL31 (glycan-conjugate-biotin) was monitored for 90 seconds. Dissociation was monitored for 600 seconds. A buffer-only blank curve was subtracted to correct for any drift. Data were fitted to a 2:1 binding model using ForteBio™ data analysis software to determine k _on , k _off , and kd. The buffer for dilution and all binding steps was 20 mM phosphate, 150 mM NaCl, pH 7.2.

Canine IL31 with a poly-His tag at the C-terminus was expressed from CHO-S cells and purified. Human IL31 was obtained from Sino Biological and the streptavidin biosensor was obtained from ForteBio (Cat. #18-509). Binding kinetics were as follows. For the ligand canine IL31 (amine-conjugate-biotin), the Kd(M) for chimeric M14 is <1.0×10 ⁻¹¹ (FIG. 2) and for caninized M14 is <1.0×10 ⁻¹¹ (FIG. 2). 3). For the ligand canine IL31 (glycan-conjugate-biotin), caninized M14 has a Kd(M) of <1.0×10 ⁻¹² and a k _off (1/s) of <1.0×10 ^{−7 .} there were.

Chimeric M14 and caninized M14 had no apparent binding signal for human IL31. Therefore Kd could not be determined.

Example 5 Demonstration that M14 Inhibits Canine IL31 Signaling Following its binding to the IL31 receptor, IL31 activates Janus kinase (Jak) 1 and Jak2 signaling molecules. Activated Jak subsequently stimulates phosphorylation of downstream signaling STAT-3 and STAT-5. Anti-phospho-Stat3 immunoblot analysis was used to detect anti-IL31 activity from Protein A-purified fractions of cell-free media (Gonzales et al., Vet Dermatol, 2013, 24, 48-e12). Briefly, canine monocyte DH82 cells (American Type Culture Collection, Manassas, VA, USA) were incubated with 15% heat-inactivated fetal bovine serum, 2 mmol/L GlutaMax, 1 mmol/L sodium pyruvate, and 10 ng/L 96-well flat bottom cell culture at a density of 1×10 ⁵ cells per well for 24 hours at 37° C. in MEM growth medium (Life Technologies) containing mL canine interferon-c (R&D Systems, Minneapolis, Minn., USA) Plates were seeded. In this experiment, the concentration of canine IL31-Fc was 5 ng/mL (8 nM). Anti-phospho-STAT-3 and anti-STAT-3 antibodies were purchased from R&D Systems. Anti-β-actin antibody was obtained from Sigma-Aldrich. As shown in FIG. 4, canine IL31 signaling decreased (as evidenced by decreased phosphorylation of STAT-3) with increasing concentrations of caninized M14 exposed to cells (lane 1: no anti-IL31 antibody, lane 2: 3.3 nM, lane 3: 6.6 nM, lane 4: 9.9 nM, and lane 5: 13.2 nM).

Example 6 Identification of M14 Canine IL31 Binding Epitopes To identify the canine IL31 epitopes recognized by M14, multiple GST canine IL31 fragment fusion molecules were generated and proteins expressed in cells of E. coli. . After transferring the GST fusion protein to the membrane, the chimeric M14 was used to probe the membrane. A positive signal was obtained when the IL31 fragment contained the epitope.

A combination of Figures 5 and 6 demonstrated that M14 could recognize the smallest fragment (SEQ ID NO:23).

Example 7: Demonstration that M14 cross-reacts with feline IL31 To determine whether the M14 antibody recognizes feline IL31 (SEQ ID NO:28) or equine IL31 (SEQ ID NO:29), the respective proteins were and expressed in mammalian 293 cells. Partially purified proteins were blotted onto membranes and probed with M14 antibody. The immunoblot in Figure 1 demonstrates that M14 binds to feline IL31. Immunoblot assays did not detect binding between M14 and equine IL31. However, biolayer interference analysis revealed that the M14 antibody binds equine IL31, albeit with low affinity. Preliminary Kd measurements using biotinylated equine IL31 immobilized on the sensor revealed an affinity (Kd) of approximately 10-50 nM.

Example 8: Feline M14
The M14 variable light chain was catified as (SEQ ID NO:32) and the M14 variable heavy chain was catified as (SEQ ID NO:33). First, mouse heavy and light chain variable sequences were used to search for correct variants of feline VH and VL. The correct cat frame was selected for grafting the CDRs. Structural modeling was used to further optimize these. The feline VH and VL were fused to the feline IgG heavy chain constant domains (CH1, CH2, and CH3) and the feline light chain constant domain (CL1).

Feline M14 chimeric antibodies (SEQ ID NOs:30 and 31) or feline M14 antibodies (SEQ ID NOs:34 and 35) can be administered to cats for the treatment of IL31-induced conditions.

Example 9 Identification of M14 Canine IL31 Binding Epitopes To further identify the amino acid residues of the canine IL31 epitope recognized by M14, multiple GST canine IL31 epitope fragment fusion molecules with alanine mutations were expressed in E. coli. Figures 8-12 show immunoblots of fine epitope mapping of canine IL31-GST fusion proteins probed with anti-canine IL31-mAb (M14) or caninized M14 (upper panel) and anti-GST antibody control (lower panel). indicates Epitope fragments tested in each lane are listed below the immunoblot. Fragment names indicate the amino acid range of mature canine IL31 (SEQ ID NO:44) tested and the position of the alanine mutation, if included. A positive signal was generated when the IL31 fragment contained the wild-type epitope, and a negative signal was generated when the IL31 fragment contained an alanine substitution at a residue important for antibody-ligand interaction.

The results of the epitope mapping studies are summarized in Table 3 below. The results indicate that P12, S13, D14, and K17 of mature canine IL31 (SEQ ID NO:44) are involved in binding antibody M14, and that R16 and I18 are partially involved in recognizing M14. suggesting. Taken together, the results of this study suggest that the IL31 polypeptide motif that binds to the CDRs _of antibody M14 is PSDX1X2KI, where _{X1 and X2} are variable (SEQ ID NO:45). ing.
Table 3.

Example 10: M14 specifically binds IL31 of other species with the _{PSDX1X2KI epitope} motif.
The IL31 epitope motif (PSDX ₁ X ₂ KI, SEQ ID NO: 45) recognized by M14 is not present in the human (SEQ ID NO: 46) or mouse (SEQ ID NO: 61) IL31 amino acid sequences. However, this motif is found in the domestic cat Felis catus (XP_011286140.1; SEQ ID NO: 28).
Walrus (Odobenus rosmarus divergens) (XP_004395998.1; SEQ ID NO: 47)
Papio Anubis (XP_003907358.1; SEQ ID NO: 49)
Polar bear (Ursus maritimus) (XP_008687166.1; SEQ ID NO: 51)
Weddell Seal (Leptonychotes weddellii) (XP_006746595.1; SEQ ID NO: 52)
Amur tiger (Panthera tigris altaica) (XP_007079636.1; SEQ ID NO: 53)
Cheetah (Acinonyx jubatus) (XP_014919275.1; SEQ ID NO: 54)
Cynomolgus monkey (Macaca fascicularis) (EHH66805.1; SEQ ID NO: 55)
Rhesus monkey (Macaca mulatta) (EHH21279.1; SEQ ID NO: 56)
Drill (Mandrillus leucophaeus) (XP_011819882.1; SEQ ID NO: 57)
Green monkey (Chlorocebus sabaeus) (XP_008003211.1; SEQ ID NO: 58)
Sooty mangabey (Cercocebus atys) (XP_011926625.1; SEQ ID NO: 59)
Golden snub-nosed monkey (Rhinopithecus roxellana) (XP_010366647.1; SEQ ID NO: 60)
have been identified in several other species, including

Walrus IL31 (SEQ ID NO:47) and Anubis baboon IL31 (SEQ ID NO:49) possess a PSDX ₁ X ₂ KI epitope (SEQ ID NO:45) recognizable by antibody M14. A C-terminal His-tag was added to walrus IL31 (SEQ ID NO: 48) and baboon IL31 (SEQ ID NO: 50) to facilitate protein purification. Western blot analysis confirmed that M14 bound to walrus IL31 (Figure 13). M14 antibodies or derivatives thereof can be used as therapeutics and diagnostics for IL31-induced diseases in any of the species listed above.

Example 11: Thermostability of Caninized M14 Antibody The thermostability of caninized M14 antibody was compared to Zoetis' CYTOPOINT™, a commercial anti-IL31 antibody, using differential scanning fluorescence (DSF) over a wide range. was analyzed over the pH of The melting temperature (Tm) for each antibody at various pHs is listed in Table 4 below. Both CYTOPOINT™ and caninized M14 antibodies were buffer exchanged into the assay buffers listed in Table 4 using PD Minitrap™ G-25 columns (GE Healthcare). The Tm of each antibody was evaluated using the same buffer and protein concentration. The caninized M14 antibody exhibited improved thermal stability compared to CYTOPOINT™ over a wide range of pH. Furthermore, under stress conditions of 0.22 mg/ml antibody at 55° C. for 2 days, CYTOPOINT™ precipitated, whereas no precipitation was observed with the caninized M14 antibody.
Table 4.

Example 12 Caninized M14 Antibody Buffer Formulations The thermal stability of caninized M14 antibody in various buffer formulations was analyzed. Buffers containing sodium phosphate, sodium acetate, or L-histidine were considered. Other formulation variables include different pH (pH 5.2, 5.5, 6.0, 6.5, and 7.0), different sodium chloride concentrations (50 mM and 140 mM), different polysorbates (polysorbate 20 and Polysorbate 80), and different antimicrobial agents (m-cresol and methylparaben) were included. The melting temperature (Tm) of caninized M14 antibody in each buffer was determined by differential scanning fluorescence (DSF) technique from 20°C to 95°C. Table 5 lists the Tm values for caninized M14 antibodies in various buffers tested.
Table 5

Further testing the Tm of formulations D1, D2, D3, D4, D5, and D6 in the presence of each one of the following sugars (1%), sucrose, trehalose, D-mannitol, maltose, and sorbitol using DSF bottom.

Formulations in the presence and absence of sugars were also tested under stress conditions of 40° C. for 1 day, then 45° C. for 1 day, then 55° C. for 4 days. Size exclusion HPLC analysis was used to detect and quantify the monomeric and aggregated forms of the antibody in various formulations after being subjected to stress conditions. Samples were loaded onto a SHODEX™ KW803 column (8 mm x 300 mm) and attached to a KW-G guard column. An Agilent 1100 chromatography system was used with 2x PBS (270 mM NaCl, 5.4 mM KCl, 8.6 mM _Na2PO4 ) as running buffer, pH _7.2 , and a constant flow rate of 0.5 mL/min. The column was calibrated using BIORAD gel filtration standards (Catalog No. 151-1901) consisting of thyroglobulin, bovine γ-globulin, chicken ovalbumin, horse myoglobin, and vitamin B12 (molecular weight 1,350-670,000). The amount of monomeric antibody remaining in solution was determined by measuring the UV absorbance at 214 nm or 280 nm and calculating the peak area under the curve.

Based on DSF and HPLC analysis, formulations such as D1, D2, D3, D4, D6, D7, D10, and D12 contain L-histidine, sodium chloride, polysorbate 80 and have a pH of 5.0-6.2. A formulation with For example, a desirable formulation for single doses is 20 mM L-histidine, 140 mM sodium chloride, polysorbate 80 (0.05 mg/mL), pH 5.5, and desirable for multiple doses (in the presence of preservatives). The formulation is
1.20 mM L-histidine, 140 mM sodium chloride, polysorbate 80 (0.05 mg/mL), sucrose (1-3%), m-cresol (0.2%), pH 5.5 and 2.20 mM L - histidine, 140 mM sodium chloride, polysorbate 80 (0.05 mg/mL), trehalose (1-3%), methylparaben (0.9%), pH 5.5
is.

<Further embodiment>
[Embodiment 1]
An isolated antibody that binds to canine IL31, wherein the antibody binds to an epitope comprising the amino acid sequence of SEQ ID NO:23.
[Embodiment 2]
An isolated antibody that binds to canine IL31, wherein the antibody binds to an epitope comprising the amino acid sequence of PSDX1X2KI ( _{SEQ ID} NO:45), wherein X is any amino acid residue.
[Embodiment 3]
X ₁ is a hydrophobic amino acid, or X ₁ is selected from A, V, I, and L, or X ₁ is selected from V and I and X ₂ is a hydrophilic amino acid, or 3. The isolated antibody of embodiment ₂ , wherein X2 is selected from A, R, K, Q and N, or X2 _is selected from R and Q.
[Embodiment 4]
The isolated antibody of embodiment 2 or embodiment 3, _wherein X1 is V and _X2 is R, or X1 _is I and _X2 is Q.
[Embodiment 5]
5. The antibody of any one of embodiments 2-4, which binds to an epitope comprising the amino acid sequence of SEQ ID NO:88.
[Embodiment 6]
Less than 5×10 ⁻⁶ M, less than 1×10 −6 M, less than 5×10 ⁻⁷ M, less than 1×10 ⁻⁷ M, less than 5×10 ^{−8 M} , 1× as measured by biolayer interferometry Less than 10 ⁻⁸ M, less than 5×10 ⁻⁹ M, less than 1×10 ⁻⁹ M, less than 5×10 ⁻¹⁰ M, less than 1×10 ⁻¹⁰ M, less than 5×10 ⁻¹¹ M, 1×10 ^{− 6.} The antibody of any one of embodiments 1-5, which binds canine IL31 with a dissociation constant (Kd) of less than 11 M, less than 5x10-12 M, or ^less than 1x10-12 ^M.
[Embodiment 7]
7. The antibody of any one of embodiments 1-6, which reduces IL31 signaling function in a companion animal species as measured by reduced phosphorylation of STAT-3.
[Embodiment 8]
8. The antibody of embodiment 7, wherein the companion animal species is canine, feline or equine.
[Embodiment 9]
9. The antibody of any one of embodiments 1-8, which binds to feline IL31 or equine IL31 as determined by immunoblot analysis and/or biolayer interferometry.
[Embodiment 10]
10. The antibody of any one of embodiments 1-9, which competes with monoclonal M14 antibody for binding to canine IL31.
[Embodiment 11]
11. The antibody of any one of embodiments 1-10, which competes with monoclonal M14 antibody for binding to feline IL31.
[Embodiment 12]
12. The antibody of any one of embodiments 1-11, which does not bind human IL31, as determined by immunoblot analysis and/or biolayer interferometry.
[Embodiment 13]
13. The antibody of any one of embodiments 1-12, which is a monoclonal antibody.
[Embodiment 14]
14. The antibody of any one of embodiments 1-13, which is a canine, canine, feline, feline, equine, equine, or chimeric antibody.
[Embodiment 15]
15. The antibody of any one of embodiments 1-14, which is a chimeric antibody comprising a murine variable heavy chain framework region or a murine variable light chain framework region.
[Embodiment 16]
comprising heavy and light chains,
a) a CDR-H1 sequence whose heavy chain has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 1; a CDR-H2 sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence; and at least 85%, at least 90%, at least 95% with the amino acid sequence of SEQ ID NO:3, or a CDR-H3 sequence with at least 98% sequence identity, and
b) a CDR-L1 sequence whose light chain has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:63; a CDR-L2 sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with; and at least 85%, at least 90%, at least 95%, or at least the amino acid sequence of SEQ ID NO:10 comprising a CDR-L3 sequence with 98% sequence identity;
16. The antibody of any one of embodiments 1-15.
[Embodiment 17]
a) (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:89; and (iii) a CDR- comprising the amino acid sequence of SEQ ID NO:3 a heavy chain comprising H3, or
b) (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:62 or SEQ ID NO:87; and (iii) a CDR- comprising the amino acid sequence of SEQ ID NO:3 Heavy chain containing H3
17. The antibody of any one of embodiments 1-16, comprising
[Embodiment 18]
a) (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:10. chain or
b) (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:63; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:10. chain
18. The antibody of any one of embodiments 1-17, comprising a
[Embodiment 19]
(a) variable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO: 4, 70 or 79; (b) HC-FR2 sequence of SEQ ID NO: 5, 71 or 80; (c) SEQ ID NO: 6, 72; (d) the HC-FR4 sequence of SEQ ID NO: 7, 74 or 82; (e) the variable region light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 11, 75 or 83; (f) LC-FR2 sequences of SEQ ID NOs: 12, 76 or 84; (g) LC-FR3 sequences of SEQ ID NOs: 13, 77 or 85; or (h) LC-FR4 sequences of SEQ ID NOs: 14, 78 or 86.
19. The antibody of any one of embodiments 16-18, further comprising one or more of:
[Embodiment 20]
a) (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:24; (ii) at least with the amino acid sequence of SEQ ID NO:25; a variable heavy chain sequence having 85%, at least 90%, at least 95%, or at least 98% sequence identity, or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or
b) (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 16; (ii) at least with the amino acid sequence of SEQ ID NO: 15; or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii), or
c) (i) a variable light chain sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:32; (ii) at least with the amino acid sequence of SEQ ID NO:33; or (iii) the variable light chain sequence of (i) and the variable heavy chain sequence of (ii).
20. The antibody of any one of embodiments 1-19, comprising a
[Embodiment 21]
21. The antibody of any one of embodiments 1-20, comprising the variable light chain sequence of SEQ ID NO:24, SEQ ID NO:16 or SEQ ID NO:32.
[Embodiment 22]
22. The antibody of any one of embodiments 1-21, comprising the variable heavy chain sequence of SEQ ID NO:25, SEQ ID NO:15 or SEQ ID NO:33.
[Embodiment 23]
The variable light chain sequence of SEQ ID NO:24 and the variable heavy chain sequence of SEQ ID NO:25, the variable light chain sequence of SEQ ID NO:16 and the variable heavy chain sequence of SEQ ID NO:15; or the variable light chain sequence of SEQ ID NO:32 and SEQ ID NO:33 23. The antibody of any one of embodiments 1-22, comprising a variable heavy chain sequence of
[Embodiment 24]
24. The antibody of any one of embodiments 1-23, which comprises a constant heavy chain region or a constant light chain region derived from a companion animal.
[Embodiment 25]
(a) a canine heavy chain constant region selected from IgG-A, IgG-B, IgG-C and IgG-D constant regions; (b) a feline heavy chain constant region selected from IgGl, IgG2a and IgG2b constant regions; or (c) the antibody of any one of embodiments 1-24, which comprises an equine heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6 and IgG7 constant regions.
[Embodiment 26]
a) (i) the light chain amino acid sequence of SEQ ID NO:26; (ii) the heavy chain amino acid sequence of SEQ ID NO:27; or (iii) the light chain amino acid sequence of (i) and the heavy chain amino acid sequence of (ii), or
b) (i) the light chain amino acid sequence of SEQ ID NO:30, (ii) the heavy chain amino acid sequence of SEQ ID NO:31, or (iii) the light chain amino acid sequence of (i) and (ii) the heavy chain amino acid sequence, or
c) (i) the light chain amino acid sequence of SEQ ID NO:34; (ii) the heavy chain amino acid sequence of SEQ ID NO:35; or (iii) the light chain amino acid sequence of (i) and the heavy chain amino acid sequence of (ii)
26. The antibody of any one of embodiments 1-25, comprising a
[Embodiment 27]
27. The antibody of any one of embodiments 1-26, comprising the light chain amino acid sequence of SEQ ID NO:21.
[Embodiment 28]
28. The antibody of any one of embodiments 1-27, comprising the heavy chain amino acid sequence of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.
[Embodiment 29]
29. The antibody of any one of embodiments 1-28, which is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab') ₂ and Fab'-SH.
[Embodiment 30]
bispecific, IL31 and one or more antigens selected from IL17, TNFα, CD20, CD19, CD25, IL4, IL13, IL23, IgE, CD11α, IL6R, α4-integrin, IL12, IL1β or BlyS 30. The antibody of any one of embodiments 1-29, which binds to.
[Embodiment 31]
An isolated nucleic acid encoding the antibody of any one of embodiments 1-30.
[Embodiment 32]
A host cell comprising a nucleic acid according to embodiment 31.
[Embodiment 33]
33. A method of producing an antibody comprising culturing the host cell of embodiment 32 and isolating the antibody.
[Embodiment 34]
31. A pharmaceutical composition comprising the antibody of any one of embodiments 1-30 and a pharmaceutically acceptable carrier.
[Embodiment 35]
A pharmaceutical composition comprising an anti-IL31 antibody and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises L-histidine, sodium chloride and polysorbate 80, and the pharmaceutical composition is 5.0- A pharmaceutical composition having a pH of 6.2.
[Embodiment 36]
36. The pharmaceutical composition according to embodiment 35, having a pH of 5.0 to 6.0, or 5.3 to 5.7, or 5.5.
[Embodiment 37]
37. The pharmaceutical composition according to embodiment 35 or embodiment 36, having an L-histidine concentration of 5 mM to 100 mM, 10 mM to 50 mM, 20 mM to 30 mM, 10 to 30 mM, or 20 mM.
[Embodiment 38]
38. The pharmaceutical composition according to any one of embodiments 35-37, having a sodium chloride concentration of 80-200 mM, 100-175 mM, 120-150 mM, or 140 mM.
[Embodiment 39]
39. Any one of embodiments 35-38, having a polysorbate 80 concentration of 0.005 mg/mL to 0.5 mg/mL, 0.01 mg/mL to 0.1 mg/mL, or 0.05 mg/mL. pharmaceutical composition.
[Embodiment 40]
40. The pharmaceutical composition according to any one of embodiments 35-39, wherein the pharmaceutically acceptable carrier comprises at least one sugar.
[Embodiment 41]
41. A pharmaceutical composition according to embodiment 40, having a sugar concentration of 0.5% to 20%, 1% to 10%, 1% to 5%, or 1% to 3%.
[Embodiment 42]
42. The pharmaceutical composition according to any one of embodiments 35-41, wherein the pharmaceutically acceptable carrier comprises sucrose, trehalose, D-mannitol, maltose and/or sorbitol.
[Embodiment 43]
43. The pharmaceutical composition according to any one of embodiments 35-42, comprising an antimicrobial agent.
[Embodiment 44]
44. The pharmaceutical composition according to any one of embodiments 35-43, comprising m-cresol or methylparaben.
[Embodiment 45]
45. The pharmaceutical composition according to any one of embodiments 35-44, comprising 0.2% m-cresol and/or 0.9% methylparaben.
[Embodiment 46]
46. A pharmaceutical composition according to any one of embodiments 35-45, wherein the anti-IL31 antibody is an antibody according to any one of embodiments 1-30.
[Embodiment 47]
A method of treating a companion animal species having an IL31-induced condition, comprising a therapeutically effective amount of an antibody according to any one of embodiments 1-30 or any one of embodiments 34-46. A method comprising administering the described pharmaceutical composition to a companion animal species.
[Embodiment 48]
48. The method of embodiment 47, wherein the companion animal species is canine, feline, or equine.
[Embodiment 49]
49. The method of embodiment 47 or embodiment 48, wherein the condition induced by IL31 is an pruritus or allergic condition.
[Embodiment 50]
50. The method of any one of embodiments 47-49, wherein the IL31-induced condition is selected from atopic dermatitis, pruritus, asthma, psoriasis, scleroderma and eczema.
[Embodiment 51]
51. The method of any one of embodiments 47-50, wherein the antibody or pharmaceutical composition is administered parenterally.
[Embodiment 52]
52. of embodiments 47-51, wherein the antibody or pharmaceutical composition is administered by an intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intrasynovial, intrathecal or inhalational route. A method according to any one of paragraphs.
[Embodiment 53]
53. The method of any one of embodiments 47-52, comprising administering a Jak inhibitor, PI3K inhibitor, AKT inhibitor or MAPK inhibitor in combination with an antibody or pharmaceutical composition.
[Embodiment 54]
anti-IL17 antibody, anti-TNFα antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD25 antibody, anti-IL4 antibody, anti-IL13 antibody, anti-IL23 antibody, anti-IgE antibody, anti-CD11α antibody, in combination with an antibody or pharmaceutical composition, 54. Any one of embodiments 47-53, comprising administering one or more antibodies selected from anti-IL6R antibodies, anti-α4-integrin antibodies, anti-IL12 antibodies, anti-IL1β antibodies, and anti-BlyS antibodies. the method of.
[Embodiment 55]
A method of reducing IL31 signaling function in a cell, wherein the antibody of any one of embodiments 1-30 or the pharmaceutical composition of any one of embodiments 34-46 is administered to an extracellular exposing the cell under conditions that allow binding of the antibody to IL31, thereby reducing binding to the IL31 receptor and/or reducing IL31 signaling function by the cell.
[Embodiment 56]
56. The method of embodiment 55, wherein the cells are exposed to the antibody or pharmaceutical composition ex vivo.
[Embodiment 57]
56. The method of embodiment 55, wherein the cells are exposed to the antibody or pharmaceutical composition in vivo.
[Embodiment 58]
58. The method of any one of embodiments 55-57, wherein the cells are canine, feline or equine cells.
[Embodiment 59]
A method for detecting IL31 in a sample from a companion animal species, comprising an antibody according to any one of embodiments 1-30 or a pharmaceutical composition according to any one of embodiments 34-46 A method comprising contacting an article with a sample under conditions that allow binding of the antibody to IL31 and detecting whether a complex is formed between the antibody and IL31 in the sample.
[Embodiment 60]
60. The method of embodiment 59, wherein the sample is a biological sample obtained from a dog, cat or horse.

Claims (27)

( i) a heavy chain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, CDR-H2 comprising the amino acid sequence of SEQ ID NO:89, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, and (ii) SEQ ID NO: a light chain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10 ;
i) a variable light chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:24; and a variable heavy chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:25. or
ii) a variable light chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:16; and a variable heavy chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:15 or
iii) a variable light chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:32; and a variable heavy chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:33
including,
An antibody that binds to canine IL-31. (i) a heavy chain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 87, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (ii) SEQ ID NO: a light chain comprising a CDR-L1 comprising the amino acid sequence of 63, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:10;
i) a variable light chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:24; and a variable heavy chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:25. or
ii) a variable light chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:16; and a variable heavy chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:15. or
iii) a variable light chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:32; and a variable heavy chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:33
including,
An antibody that binds to canine IL-31. 3. The antibody of claim 1 or 2, which is a monoclonal antibody. 4. The antibody of any one of claims 1-3, which is a caninized antibody, a feline antibody, an equine antibody, or a chimeric antibody. 5. The antibody of any one of claims 1-4, comprising a variable light chain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:24, SEQ ID NO:16 or SEQ ID NO:32. 6. The antibody of any one of claims 1-5, comprising a variable light chain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:25, SEQ ID NO:15 or SEQ ID NO:33. a variable light chain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:24 and a variable heavy chain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:25;
a variable light chain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 16 and a variable heavy chain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 15; or
A variable light chain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:32 and a variable heavy chain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:33
7. The antibody of any one of claims 1-6, comprising 5. The antibody of any one of claims 1 , 3 or 4 , comprising a variable light chain comprising the amino acid sequence of SEQ ID NO:24, SEQ ID NO:16 or SEQ ID NO:32. 9. The antibody of any one of claims 1 , 3, 4 or 8 , comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:25, SEQ ID NO:15 or SEQ ID NO:33. a variable light chain comprising the amino acid sequence of SEQ ID NO:24 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:25; a variable light chain comprising the amino acid sequence of SEQ ID NO:16 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:15; or sequences 10. The antibody of any one of claims 1 , 3, 4, 8 or 9 , comprising a variable light chain comprising the amino acid sequence of SEQ ID NO:32 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:33. (a) canine heavy chain constant regions selected from IgG-A, IgG-B, IgG-C and IgG-D constant regions; (b) feline heavy chain constant regions selected from IgGl, IgG2a and IgG2b constant regions; or (c) an equine heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4 , IgG5, IgG6 and IgG7 constant regions. a) (i) a light chain comprising the amino acid sequence of SEQ ID NO:26, (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO:27, or (iii) a light chain of (i) and a heavy chain of (ii); ) (i) a light chain comprising the amino acid sequence of SEQ ID NO:30, (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO:31, or (iii) the light chain of (i) and the heavy chain of (ii); or c) (i) a light chain comprising the amino acid sequence of SEQ ID NO:34, (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO:35, or (iii) the light chain of (i) and the heavy chain of (ii); or d) ( i) a light chain comprising the amino acid sequence of SEQ ID NO: 21, (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, or (iii) the light chain of (i) ( 12. The antibody of any one of claims 1 , 3, 4 or 8-11 , comprising the heavy chain of ii). 11. The antibody of any one of claims 1-10 , which is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab') ₂ and Fab'-SH. multispecific and one or more antigens selected from IL31 and IL17, TNFα, CD20, CD19, CD25, IL4, IL13, IL23, IgE, CD11α, IL6R, α4-integrin, IL12, IL1β or BlyS 14. The antibody of any one of claims 1-13 , which binds to. 15. An isolated nucleic acid encoding the antibody of any one of claims 1-14 . A host cell comprising the nucleic acid of claim 15 . 17. A method of producing an antibody comprising culturing the host cell of claim 16 and isolating the antibody. 15. A pharmaceutical composition comprising the antibody of any one of claims 1-14 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the anti-IL - 31 antibody of any one of claims 1 to 14 and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is L-histidine, sodium chloride and polysorbate 80, wherein the pharmaceutical composition has a pH of 5.0 to 6.2. 20. A method of treating a companion animal species having an IL31-induced condition, comprising a therapeutically effective amount of an antibody according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 18 or 19 . A method comprising administering an agent to a companion animal species. 21. The method of claim 20 , wherein the companion animal species is canine, feline, or equine. the condition induced by IL31 is an pruritus or allergic condition, or
the IL31-induced condition is selected from atopic dermatitis, pruritus, asthma, psoriasis, scleroderma and eczema;
22. A method according to claim 20 or 21 . the antibody or pharmaceutical composition is administered parenterally, and/or
23. The antibody or pharmaceutical composition of claims 20-22 , wherein the antibody or pharmaceutical composition is administered by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intrasynovial, intrathecal or inhalation route. A method according to any one of paragraphs. in combination with the antibody or pharmaceutical composition,
(i) Jak inhibitors, PI3K inhibitors, AKT inhibitors or MAPK inhibitors, and/or (ii) anti-IL17 antibodies, anti-TNFα antibodies, anti-CD20 antibodies, anti-CD19 antibodies, anti-CD25 antibodies, anti-IL4 antibodies, anti- administering one or more antibodies selected from IL13 antibody, anti-IL23 antibody, anti-IgE antibody, anti-CD11α antibody, anti-IL6R antibody, anti-α4-integrin antibody, anti-IL12 antibody, anti-IL1β antibody, and anti-BlyS antibody 24. The method of any one of claims 20-23 , comprising 20. A method of reducing IL31 signaling function in a cell, comprising administering an antibody according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 18 or 19 ex vivo or in vivo, exposing the cell under conditions that allow binding of the antibody to extracellular IL31, thereby reducing binding to the IL31 receptor and/or reducing IL31 signaling function by the cell can be,
A method wherein the cells are canine, feline or equine cells. 20. A method for detecting IL31 in a sample from a companion animal species, comprising administering an antibody according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 18 or 19 to IL31. contacting the sample under conditions that allow binding of the antibody of , and detecting whether a complex is formed between the antibody and IL31 in the sample. 27. The method of claim 26 , wherein the sample is a biological sample obtained from a dog, cat or horse.

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